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

Riepilogo

This protocol presents a step-by-step methodological approach of exposing bone metastatic cells lines and primary bone tumors to 5-aminolevulinic acid-mediated photodynamic therapy (PDT). The effects on cell migration potential/invasiveness, viability, apoptosis, and senescence potential are also analyzed following PDT exposure.

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.

Introduzione

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.

Protocollo

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.

Risultati

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 ...

Discussione

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 activat...

Materiali

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