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JoVE Journal Cancer Research

Cancer Research

5-Aminolevulinic Acid-mediated Photodynamic Therapy on Primary Bone Tumor and Bone Metastases Cell Lines

Published: May 10, 2022 doi: 10.3791/63644
*1,2, *2, 1,2, 1, 1,2, 1
1Laboratory of Cell Biology, Department of Orthopaedic Surgery, University Hospital of Tübingen, 2Department of Orthopaedic Surgery, University Hospital of Tübingen
* These authors contributed equally

Abstract

Bone metastases are associated with poor prognosis and low quality of life for the affected patients. Photodynamic therapy (PDT) emerges as a noninvasive therapy that can target local metastatic bone lesions. This paper presents an in vitro method to study the PDT effect in adherent cell lines. To this end, we demonstrate a step-by-step approach to subject both primary (giant cell bone tumor) and human bone metastatic cancer cell lines (derived from a primary invasive ductal breast carcinoma and renal carcinoma) to 5-aminolevulinic acid (5-ALA)-mediated PDT.

After 24 h post 5-ALA-PDT irradiation (blue light-wavelength 436 nm), the therapeutic effect was assessed in terms of cell migration potential, viability, apoptotic features, and cellular growth arrest (senescence). Post 5-ALA-PDT irradiation, musculoskeletal-derived cell lines respond differently to the same doses and exposure of PDT. Depending on the extent of cellular damage triggered by PDT exposure, two different cell fates-apoptosis and senescence were noted. Variable sensitivity to PDT therapy among different bone cancer cell lines provides useful information for selecting more appropriate PDT settings in clinical settings. This protocol is designed to exemplify the use of PDT in the context of musculoskeletal neoplastic cell lines. It may be adjusted to investigate the therapeutic effect of PDT on various cancer cell lines and various photosensitizers and light sources.

Introduction

Therapeutic options for bone metastases are still limited and challenging despite ongoing developments in oncological treatment. The current standard method is radiotherapy, which is associated with complications such as local erythema, toxicity to inner organs1, and insufficient fractures2. There is a need for alternative antineoplastic therapies as patients with bone metastases often suffer from pain, hypercalcemia, and neurological symptoms that result in impaired mobility and reduced quality of life3. Recent findings demonstrate that PDT provides a promising, alternative, antineoplastic treatment option to directly target bone lesions, which can be used alone or supportively to radiotherapy4.

The mechanism of PDT is essentially based on an energy transfer from a light-excited photosensitive compound (photosensitizer) to tissue oxygen. This photosensitizer works similarly to a capacitor on a nanoscopic level. It can store energy in a ground state when irradiated with an appropriate wavelength of light and releases stored energy when it returns from an excited state to the original ground state5. The released energy leads to two photochemical reactions: one is the transformation of oxygen to reactive oxygen radicals by transferring hydrogen or an electron. The second is the production of singlet oxygen particles by horizontal energy transfer from the photosensitizer substrate to local triplet oxygen particles6. Reactive oxygen radicals and singlet oxygen molecules have highly cytotoxic effects on local tumor cells and induce vascular occlusion and local inflammatory response by apoptosis of endothelial cells of tumor blood vessels7.

Conventional photosensitizers are derivatives of the porphyrin family such as hematoporphyrins and benzoporphyrins8. Applying photosensitizer substances with higher affinity to tumor tissue can increase the selectivity of PDT9 y. In particular, 5-ALA, which is a biosynthetic precursor of protoporphyrin IX, can accumulate in tumor cells such as actinic keratosis, basal cell carcinoma, bladder tumor, and gastrointestinal cancer5. Different delivery approaches using 5-ALA can also vary the efficiency of PDT in relation to tumor localization. Thus, topical use of 5-ALA with the application of PDT became the first-line dermatologic therapy against actinic keratosis10. Recent results for bone metastases of invasive ductal breast cancer cell lines indicate possible inhibition of cell migration and induction of apoptosis after exposure to PDT with 5-ALA11. However, using PDT in subfascial human tissue such as bone tissue is still in its preclinical to experimental clinical stage as the efficacy needs to be improved. Applications of nanoparticles with light-based therapy already show great impact in dentistry12. Thus, it is likely that combining the use of nanoparticles with PDT will expand its application range towards orthopedic oncology.

The following protocol describes how to prepare both cells originating from primary bone tumors and bone metastases cell lines and subject them to 5-ALA-mediated PDT for a predefined time exposure. A detailed description of how to perform and assess the cellular migration potential, vitality, and senescence post 5-ALA-PDT irradiation is also included. Step-by-step instructions provide a straightforward and concise approach to acquire reliable and reproducible data. The advantages, limitations, and future perspectives of the PDT approach for bone neoplastic lesions are also discussed.

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Protocol

Three different types of cell lines were employed: "MAM"-a cell line originating from bone metastases of renal cell carcinoma, "MAC"-bone metastases of an invasive ductal breast carcinoma, and "17-1012"-a giant cell tumor of bone. Marrow-derived mesenchymal stem cells (MSCs) were used as a control group. Institutional and ethical approval was obtained before the commencement of the study (project number: 008/2014BO2-for the cancer cell lines and project number: 401/2013 BO2 for MSCs).

1. Cell culture

NOTE: Culture media can be prepared beforehand. The culture medium for MAM and 17-1012 consists of RPMI supplemented with 10% (v/v) fetal bovine serum (FBS) and 2 mM L-glutamine. The culture medium for MAC and MSCs consists of Dulbecco's modified Eagle's medium (DMEM) with glutamine substitute (see the Table of Materials), 4.5 g/L D-glucose supplemented with 10% (v/v) FBS.

  1. Harvest and passage cells until 80% confluence is achieved.
  2. Wash the cells with 5 mL of phosphate-buffered saline (PBS) and aspirate the PBS from the flask using a pipette.
    NOTE: This step ensures the removal of residual complete medium containing protease inhibitors.
  3. Add 3 mL of 0.05% (v/v) trypsin-EDTA to dissociate the adherent cells from the bottom of the flask in which they are cultured.
  4. Incubate the flasks for 5 min at 37 °C in a humidified atmosphere with 5% CO2.
  5. Observe the cell detachment under a phase-contrast microscope.
    NOTE: The incubation time must be adjusted for each cell type. Prevent cell exposure to trypsin solution for longer periods (>10 min).
  6. Stop the trypsinization by adding add 3 mL of culture medium.
    NOTE: Make sure to add the appropriate culture medium for each cell type containing 10% (v/v) FBS.
  7. Transfer the detached cells to a centrifugation tube and centrifuge them for 7 min at 350 × g at 7 °C.
  8. Discard the supernatant.
  9. Resuspend the cells pellet in 1 mL of culture medium and count the cells using a hemocytometer as described previously13.

2. PDT setup and exposure

  1. Prepare the PDT device (see the Table of Materials) by plugging in all the cables and accessories.
  2. Dim the light in the room to avoid unnecessary light dissipation.
  3. Position and stabilize the light fiber with the aid of duct tape directly on top of the well plate.
    NOTE: A uniform distribution of the light fibers on top of the wells is desired to ensure that all cells are irradiated and receive the same amount of energy (Figure 1).
  4. Make sure not to severely bend or damage the light fiber as this might lead to a reduction in the light intensity.
  5. Cover the well plates with aluminum foil to minimize light dissipation.
  6. Switch on the PDT device by pressing the ON/OFF switch button.
  7. Insert the metered card into the slot in the front of the PDT device.
    NOTE: One metered card is provided with the device corresponding to 300 s exposure time. An additional 2,000 s program is automatically integrated within the PDT device. For both exposure times, the light is delivered in a continuous output mode for the indicated periods.
  8. Ensure the timer on the PDT lightbox display changes to the prescribed time indicated on the metered card.
  9. Start the PDT treatment by pressing the incorporated foot pedal.
    ​NOTE: An automatic stop function is incorporated into the device, and the system runs automatically. Do not stop the light process or remove the light fiber prior to the completion of the cycle. At the completion of the light cycle, the light source shutter is closed, and no further light is delivered.

3. Migration assay

  1. Seed cells as follows: 2 × 104 cells - MAC, 1 × 104 cells - MAM, 3 x 104 - 17-1012, and 2.5 × 104 - MSCs into each 2-chamber culture insert integrated into opaque F-bottom, 6-well plates.
  2. Incubate the cells at 37 °C, 5% CO2 for 24 h. Remove the inserts afterward.
    NOTE: To have a starting reference point for the migration and the initial unbiased gap size, a separate plate, which will not be subjected to further 5-ALA-PDT exposure (steps 3.3-3.4) but instead directly stained and quantified (steps 3.5-3.12), is employed.
  3. Replace the medium with fresh FBS-free medium containing 1 mM of 5-ALA.
  4. Incubate the cells at 37 °C, 5% CO2 for 4 h.
    NOTE: As controls, additional wells should concomitantly be covered with medium without 5-ALA.
  5. Subject the cells to PDT with blue light (436 nm, 36 J/cm2) in a continuous output mode for predefined time frames of 300 s or 2,000 s, as indicated in section 2.
    NOTE: Plates that are not subjected to PDT exposure should be employed as controls.
  6. Incubate the cells at 37 °C, 5% CO2 for 24 h.
    NOTE: Check the cells periodically to assess the degree of migration for each cell type. This must be optimized for each cell line.
  7. Wash the cells with 1.5 mL of PBS and completely remove the PBS afterward.
  8. Fix the cells by adding and incubating the cells with 1.5 mL of 4% (v/v) paraformaldehyde in PBS for 10 min.
  9. Add 1.5 mL of 70% (v/v) ethanol and incubate for 10 min.
  10. Discard the ethanol and allow the plates to air-dry at room temperature for 10 min.
  11. Incubate the cells with 1.5 mL of 0.2% (v/v) Coomassie blue dye in 90% (v/v) ethanol for 20 min.
  12. Wash the plates with double-distilled water.
    NOTE: To thoroughly wash the plates and remove the remaining debris, fully immerse the plates 2-3 times in clean, double-distilled water.
  13. Allow the plates to air-dry at room temperature for 24 h.
    NOTE: At this stage, the dried plates may be stored at room temperature for at least several weeks.
  14. Visualize and photograph the migration of cells into gaps with an inverse phase-contrast microscope at 10-fold magnification.
    NOTE: Observe and photograph the migration of live cells for up to 24 h after 5-ALA-PDT exposure. However, fixed cells can be stored and photographed later.
  15. Export the images in the desired format and quantify the gaps using software for processing and analyzing scientific images as described previously14.

4. Viability assay

  1. Seed all the cells at a density of 1.5 × 104 in 96-well plates in 50 µL of culture medium.
  2. Incubate the cells at 37 °C, 5% CO2 for 24 h in a standard cell culture incubator.
  3. Add an additional 50 µL of FBS-free culture medium containing 5-ALA at a final concentration of 1 mM.
  4. Incubate the cells at 37 °C, 5% CO2 for 4 h in a standard cell culture incubator.
    NOTE: Use 2x concentration as the cells are already covered with 50 µL of culture medium. A control with culture medium should be included.
  5. Subject the cells to PDT exposure with blue light (436 nm, 36 J/cm2) in a continuous output mode for predefined time frames of 300 s or 2,000 s, as indicated in section 2.
    NOTE: A negative control that is not subjected to PDT exposure should also be included.
  6. Incubate the irradiated cells at 37 °C, 5% CO2 for 24 h in a standard cell culture incubator.
  7. Add 15 µL of MTS reagent onto the cells and incubate the cells for 90 min at 37 °C, 5% CO2.
  8. Measure the absorbance with a spectrophotometer reader at a wavelength of 490 nm.

5. Cellular growth arrest/senescence assay (β-Galactosidase( β-Gal) activity)

NOTE: All reagents and buffers used here were provided in the assay kit (see the Table of Materials).

  1. Seed the cells at a density of 1.5 × 104 in 96-well plates in 100 µL of culture medium. Incubate the cells for 24 h, 37 °C, 5% CO2 in a standard cell culture incubator.
  2. Add 100 µL of fresh FBS-free medium with 5-ALA at a final concentration of 1 mM and incubate for 4 h at 37 °C, 5% CO2.
    NOTE: As controls, separate wells should concomitantly be covered with fresh medium without 5-ALA photosensitizer.
  3. Subject the cells to PDT exposure with blue light (436 nm, 36 J/cm2) in a continuous output mode for predefined time frames of 300 s or 2,000 s, as indicated in section 2.
    NOTE: A negative control plate that is not exposed to PDT should also be included.
  4. Incubate the cells for 24 h at 37 °C, 5% CO2 in a standard cell culture incubator.
  5. Discard the medium and wash the cells two times with PBS.
  6. Cover the cells with 100 µL of 1x cell lysis buffer and incubate for 5 min at 4 °C.
  7. Centrifuge the lysate at 350 × g 1 for 10 min at 4 °C and collect the supernatant.
  8. Pipette 50 µL of the supernatant to a new 96-well plate and add another 50 µL of 2x assay buffer.
  9. Place the new 96-well plate in a 37 °C incubator for 1.5 h.
    NOTE: The pates should be protected from light to avoid photobleaching.
  10. Transfer 50 µL of the mixture into an opaque-walled 96-well plate and add 200 µL of stop solution.
  11. Measure the absorbance with a fluorescence microplate reader at 360 nm excitation/465 nm emission.

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Representative Results

Following 5-ALA PDT exposure, the MSC-control group showed no notable effect in terms of migration following 5-ALA PDT irradiation (Figure 2A, i, v, ix). In contrast, MAC cells (Figure 1B and Figure 2A, iii, vii, xi) and 17-1012 (Figure 1B and Figure 2A, ii, vi, x) cells exhibited a decrease in migration potential for both 300 s or 2,000 s light exposure. In contrast, no notable effect in terms of migration potential was observed for the MAM cell line (derived from bone metastases of renal carcinoma). However, a decrease in viability was observed for the MAC cell line (p = 0.009) at a 2,000 s exposure time. In contrast, the 17-1012 cell line showed an increasing tendency (p = 0.007) at an exposure of 300 s, followed by a decrease (p = 0.257, Figure 3) at 2,000 s.

In contrast, a constant increase in viability was observed for both exposure times (p = 0.022, 2,000 s, Figure 3) for the MAM cell line. The control group, MSCs, maintained their viability irrespective of the exposure time (Figure 3). With respect to the cellular growth arrest (senescence), higher β-Gal activity was observed for the MAM cell line and MSCs (control group) than the MAC and 17-1012 cell lines (Figure 4).

Collectively, these results showed that human cell lines have distinct and individual 5-ALA-PDT-sensitivities. 5-ALA-PDT irradiation successfully impaired the migration potential and cellular viability of giant cell tumor of bone (17-1012) and bone metastases of invasive ductal carcinoma (MAC) cell lines. No notable effect was noted for cells derived from the bone metastases of renal carcinoma (MAM) and MSC group11.

Figure 1
Figure 1: Stepwise workflow of the experimental approach. (A) Phase-contrast images of the bone metastatic cell lines: MAC-bone metastases of invasive ductal breast carcinoma; MAM-bone metastases of renal cell carcinoma and one primary cell line 17-1012-giant cell tumor of bone; human MSCs employed as a control group throughout the experimental analysis. Images were recorded at a 10-fold magnification. Scale bars = 100 µm. (B) The cells were seeded and subjected to 1 mM 5-ALA-mediated PDT for predefined exposure times of 300 s or 2,000 s. (C) Post PDT irradiation, the cellular migration potential (10-fold magnification, scale bar = 300 µm), viability (40-fold magnification, scale bar = 50 µm), and senescence (10-fold magnification, scale bar = 300 µm) was assessed. Abbreviations: PDT = photodynamic therapy; 5-ALA = 5-aminolevulinic acid; MSCs = mesenchymal stem cells. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Cellular migration assessment following PDT exposure. (A) All cell lines (17-1012-giant cell tumor of bone (ii, vi, x); MAC-bone metastases of invasive ductal breast carcinoma (iii, vii, xi); MAM-bone metastases of renal cell carcinoma (iv, viii, xii); MSCs as a control (i, v, ix) were subjected to PDT exposure for predefined timeframes of either 300 s or 2,000 s with 5-ALA as a photosensitizer at a concentration of 1 mM. Phase-contrast microscopy representative images of cellular migration potential at 10-fold magnification. Scale bars = 300 µm. (B) Semiquantitative assessment of cellular migration potential following PDT exposure. The rate of cell migration for all cell lines was measured as a distance (µm, using the open-source software ImageJ). This figure is adapted from 11. Abbreviations: 5-ALA = 5-aminolevulinic acid; PDT = photodynamic therapy; MSCs = mesenchymal stem cells. Please click here to view a larger version of this figure.

Figure 3
Figure 3: 5-ALA PDT effect on viability of bone metastases cell lines. Boxplots depicting the viability of cells: 17-1012-giant cell tumor of bone, MAC-bone metastases of invasive ductal breast carcinoma, MAM-bone metastases of renal cell carcinoma, and MSCs as a control) previously subjected to PDT exposure for predefined time frames of 300 s or 2,000 s. Statistical comparisons were in relation to the control group (cells that were not subjected to PDT exposure). Data of 3 experiments performed in triplicates. *p < 0.05,**p < 0.01. This figure is adapted from 11. Abbreviations: 5-ALA = 5-aminolevulinic acid, O.D. = optical density; PDT = photodynamic therapy; MSCs = mesenchymal stem cells. Please click here to view a larger version of this figure.

Figure 4
Figure 4: 5-ALA PDT effect on cellular arrest (senescence) of bone metastases cell lines. Boxplots depicting cellular senescence: 17-1012-giant cell tumor of bone, MAC-bone metastases of invasive ductal breast carcinoma, MAM-bone metastases of renal cell carcinoma, and MSCs as a control) previously subjected to PDT exposure for predefined time frames of 300 s or 2,000 s. This figure is adapted from 11. Abbreviations: 5-ALA = 5-aminolevulinic acid, RFU = relative fluorescence units; PDT = photodynamic therapy; MSCs = mesenchymal stem cells; SA-β-gal = senescence-associated betagalactosidase. Please click here to view a larger version of this figure.

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Discussion

Despite current treatment options, cancer therapeutic response is variable, advocating in favor of novel approaches or even combination therapies to treat bone metastases while preserving the initial tissue structure. In this context, PDT is a promising alternative. From a simplistic point of view, PDT is comprised of two basic components: (1) a nontoxic light-sensitive dye termed photosensitizer (PS) and (2) an external light source of the appropriate wavelength that matches the absorption spectrum of the PS and activates it15. In this study, we investigated the effect of 5-ALA-PDT exposure on metastatic and primary bone tumor cell lines11.

Overall, variable sensitivity to PDT therapy was observed among different cancer bone cell lines. While 1 mM 5-ALA PDT exposure impaired the migration potential and cellular viability of bone metastases of invasive ductal carcinoma, no significant effect in terms of migration potential was observed for the giant cell tumor of bone. Further, no notable effect was observed for the bone metastases of renal carcinoma or the MSC group11. The decrease was more enhanced with a longer PDT exposure time, indicative of an exposure-dependent effect consistent with the previous research16,17. Even though accumulating evidence suggests that PDT is an efficient inducer of apoptosis in many cancer cell lines, programmed cell death is a response to overwhelming stress, whereas less severe damage initiates cellular growth arrest (senescence).

Thus, drawing an analogy to the standard of care for cancer treatment, chemotherapy, doxorubicin leads to senescence at low doses and apoptosis at high doses in breast cancer cells18. This might also apply to PDT, where light doses must be properly chosen and adapted to photo-inactivate the 5 ALA-PDT-induced senescent cells. Additionally, although we present an in vitro approach to 5-ALA PDT irradiation of metastatic and primary bone cancer cell lines11, it has to be borne in mind that only three independent experiments were conducted for each cell line. Although a sample size that is too small reduces the power of the study, preventing the findings from being extrapolated19, the measured tendency should not be affected. Cellular heterogeneity in tumors is a well-established phenomenon20,21, primarily responsible for drug resistance and treatment failure22,23. Because we analyzed only one cell line in this study, these results may not be representative of the entire neoplastic cell population but rather of a specific subclone population that exhibits common phenotypic features. In line with previous studies that showed differential sensitivity to PDT irradiation in various cancer-derived lines24,25,26, these results also showed a differential susceptibility of cancerous musculoskeletal cell lines to ALA-PDT exposure.

PDT is only minimally invasive, easy to perform, moderately effective, and allows the stereotactic placement of light fibers directly onto or in the near vicinity of tumor lesions27. Another noteworthy asset that needs to be spelled out is the uncomplicated handling of the setup. It is typically maneuvered by an external "ON/OFF" switch that controls the tumor tissues irradiation in vivo28, a critically important point in a clinical setup. A phase I clinical trial has demonstrated that the combination of vertebral metastases PDT and vertebral cement augmentation is safe from a pharmaceutical perspective and highly effective in pain reduction29. A homogeneous and reproducible light delivery rate during PDT is a determinant factor for preventing under- or overtreatment30. Flexibility is another important factor as the applied light dose must be adjustable during treatment, especially on complex body surfaces. The PDT system employed in this study employed a thin, flexible 183 cm long cylindrical shape that enabled an easy distribution of light.

For deep-seated neoplastic entities such as bone tumors, the restricted light penetration associated with PDT exposure, hypoxia, and inadequate accumulation of photosensitizer inside the tumor severely hinder the therapeutic effect of PDT31. Recent advancements in nanotechnology, such as photosensitizers encapsulated in nanoparticles, and approaches to treat deep-seated tumor sites, such as two-photon excitation, chemiluminescence, lamp implantation, and X-ray-activated photosensitizers, are gaining momentum32. Self-illuminating nanoparticles custom-designed as PDT agents-excited by enzyme-mediated bioluminescence approaches-have also been proposed33. The actual efficacy of PDT irradiations is also highly dependent on the employed light energy, tissue oxygen supply, and the optical features of the targeted tissue. However, there are several different FDA-approved PS agents34 and light sources35 available on the market36 that can be selected and adjusted according to the targeted tissue/origin.

To date, no explicit contraindications exist with respect to the combined approach of PDT with radiotherapy, radiofrequency ablation, or surgical intervention36. Moreover, the combination of immunotherapy and immunocyte-based drug delivery systems to strengthen current cancer treatment options, such as chemotherapy, PDT, and immunotherapy, may improve the effectiveness of the treatment of deep-seated tumors such as bone metastases32. In fact, for musculoskeletal neoplastic lesions, PDT has been previously shown to be successful in destroying vertebral osteolytic tumors while concomitantly enhancing vertebral structure (when combined with bisphosphonates)37.

Considering all the above-mentioned considerations, the consistency and reliability of the results post 5-ALA-PDT irradiation strongly depend on several factors: concentration of the PS, exposure time, light source, and tumor heterogeneity38. When accounting for all the aspects that may influence the overall output post PDT, the values reported in this study cannot be generalized and are rather specific for this experimental setup.

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Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgments

We thank our co-authors from the original publications for their help and support.

Materials

Name Company Catalog Number Comments
300 s metered card for PDT IlluminOss Medical Inc., East Providence, Rhode Insland, USA n/a http://www.illuminoss.com
5-aminolevulinic acid (5-ALA) photosensitizer Sigma-Aldrich, St. Louis, Missouri, USA A7793 10 mg
6 Well plates Greiner Bio-One, Frickenhausen, Germany 657160
8 Well Chamber Slides SARSTEDT AG & Co. KG, Munich, Germany 94.6140.802
96 Well plates (F-buttom) Greiner Bio-One, Frickenhausen, Germany 655180
CellTiter 96 Aqueous One
Solution Cell Proliferation
Assay (MTS-Assay)		 Promega, Fitchburg,
Wisconsin, USA		 G3580
Cellular Senescence Assay Biotrend Chemikalien GmbH, Köln, Germany CBA-231 Quantitative senescence-associated ß-galactosidase assay
Coomassie Brilliant Blue R250 Sigma-Aldrich, St Louis, Missouri, USA 35055 0.5% (w/v)
Culture-Inserts 2Well ibidi GmbH, Gräfelfing, Germany 80209
DMEM (1x) + GlutaMax-I Life Technologies, Carlsbad, Kalifornien, USA 31966-021
Fetal bovine serum (FBS) Sigma-Aldrich, St Louis, Missouri, USA F7524
Fluorescence microplate reader Promega, Madison, Wisconsin, USA GlowMAx®,
GM3510
Hemocytometer Hecht Assistent, Sondheim, Deutschland 4042
ImageJ National Institutes of Health, Be-thesda, Maryland, USA ImageJ (version: 1.53a) Software for processing and analyzing scientific images; https://imagej.net/
Inverse phase-contrast microscope Leica, Wetzlar, Germany DM IMBRE 100
Methanol AnulaR Normapur VWR, Fontenay-Sous-Bois, France 20847.307
Paraformaldehyd Sigma-Aldrich, St Louis, Missouri, USA 158127 Powder, 95% purity
PDT device (light box and accesories) IlluminOss Medical Inc., East Providence, Rhode Insland, USA n/a Blue light 436 nm, 36 J/cm2 http://www.illuminoss.com
Penicillin-Streptomycin Thermo Fisher Scientific, Waltham, Massachusetts, USA 15140-122 10,000 U/mL Penicillin
10,000 μg/mL Streptomycin
Phosphate-buffered saline (PBS) Thermo Fisher Scientific, Waltham, Massachusetts, USA 10010-015
RPMI 1640 Thermo Fisher Scientific, Waltham, Massachusetts, USA 21875034
Spectrophotomete/ microplate reader BioTek Instruments GmbH, Bad Friedrichshall, Germany EL800
Trypan Blue dye 0.4% Sigma-Aldrich, St Louis, Missouri, USA T8154
Trypsin-EDTA 10x Sigma-Aldrich, St Louis, Missouri, USA T4174

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5-aminolevulinic Acid Photodynamic Therapy Bone Tumor Bone Metastases Cell Lines Primary Invasive Ductal Breast Carcinoma Renal Carcinoma 5-ALA-PDT Blue Light Cell Migration Potential Viability Apoptotic Features Cellular Growth Arrest Senescence Musculoskeletal-derived Cell Lines Cellular Damage Apoptosis Clinical Settings Musculoskeletal Neoplastic Cell Lines
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Sachsenmaier, S. M., Walter, C.,… More

Sachsenmaier, S. M., Walter, C., Liang, C., Riester, R., Wülker, N., Danalache, M. 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Primary Bone Tumor and Bone Metastases Cell Lines. J. Vis. Exp. (183), e63644, doi:10.3791/63644 (2022).

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