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

Summary

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.

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.

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). <p class…

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 …

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 activat…

Materials

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