BSc(Chem)USA,BSc(Biol)USA,GCP Cert(London), PGCertTMed(Edinburgh), PGDipTMed(Edinburgh), MScTMed(Edinburgh),M/D Prof (London), cDSc(London),PGCertFMed(Glasgow),MRSC(Cambridge).
President and Chief Medical Editor of the Official Journal of Hellenic and International Society of Molecular and Genomic Medicine and Research.
President of the International association of Personalised Perioperative Medicine and Laser,Robotic and Genomic NANOSURGERY.

Introduction

Approximately 15-20% of breast Ca consists of triple-negative phenotype/basal-like Ca (BL/Triple(-)BCa) due to a lack of expression in ER,PR and HER-2 oncogene making it an extremely heterogeneous group of cancers that are very aggressive and have been associated with a very poor prognosis.
These tumors are characterized by an enhanced relapse pattern,and potent chemo/radioresistance due to breast CD44+ CD24- cancer stem cells (BCSCs) which are associated with cell-invasion and metastasis of breast Ca because CD44 is a receptor for hyaluronic acid that interacts with other ligands such as osteopontin which activates PI3K/AKT,HIF-1a and MMP2-9 [1],collagen that activates N-cadherin and c-JunNH(2)-terminal kinase (JNK) [2],and MMP 2,9 and 13 which dissolves bone matrix promoting osteolytic bone metastasis by complementing the activity of MMP-9,MT1-MMP and other invasive and metastatic enzymes [3]. This tumor has the tendency to metastasize to the liver,lung,bones and brain due to a mechanism which is mediated by the cell surface transmembrane glycoprotein,CD44.

More than 85% of breast cancer patients with BRCA1 mutations are triple negative tumors which are characterized by an enhanced risk of recurrence. Also,this risk is greater for cancer patients who are initially diagnosed before age forty because their tumours are much more aggressive than the tumours of older patients.Furthermore,African American breast cancer patients are at higher risk for tumour recurrence or relapse.
The reason that all the therapeutic approaches in advanced basal-like/triple(-) breast Ca such as chemotherapy,hormone-therapy,radiation,biological-therapy  and even surgery lead to cancer recurrence or relapse and metastasis is due to a small subset of cells within the tumor,the cancer stem cells (CSCs) which cause potent chemoresistance and radioresistance due to upregulated genes which activate proteins that act as drug-efflux pumps such as MDR1(ABCB1), ABCB5, MRP1(ABCC1),BCRP(ABCG2) etc [4],upregulated antiapoptotic genes, activated detoxifying or repair enzymes,and genes influencing dormancy, vascular-niche, hypoxic-stability,enhanced levels of radical scavengers, and redistribution in the cell-cycle [5] leading to self-renewal, differentiation and regeneration [6].
Breast cancer stem cells which represent only 1-2% of the total tumour cell population generate extremely heterogeneous tumour cell types because they arise from cells that possess properties of adult stem cells which self-renew rapidly and differentiate into multiple cell types which are characterized by an activated Notch signaling pathway [7].

     
Generally, conventional anticancer treatments such as chemotherapy,hormonal-therapy and even  biological-therapy cannot replace surgery which aims to remove all the cancerous tissue by using the most direct approach including lumpectomy or lump-removal, total or simple-mastectomy,modified radical-mastectomy,radical-mastectomy and some newer mastectomy procedures such as skin-sparing mastectomy, subcutaneous-mastectomy and nipple-sparing mastectomy.However,there is no difference in numbers of life-threatening metastases caused by cancer stem cells (CSCs) between lumpectomy and mastectomy with subsequent no difference in life-expectancy between these two surgical procedures.More than thirty percent of breast Ca patients that had lumpectomy required additional breast surgery or re-excision. Even with complete mastectomy,there still remains the risk of breast cancer recurrence and metastasis because this surgical procedure removes approximately 98% of the breast tissue leaving in the remaining area of 2% many breast cancer stem cells (BCSCs). 

Unfortunately even with adjuvant treatment recurrence occurs which is a cancer that returns in or near the original location.This tumour which is a derivative of breast cancer stem cells is much more aggressive,invasive,chemo/radioresistant and metastatic than the original one.These cancer stem cells are too small to be detected during treatment because MRI and CT-scans may detect tumour lesions above 1 cm while PET-scan may detect via metabolic activity facilitated by FDG absorption tumours which are larger than 0.50 cm.Thus,these cells continue to multiply growing to tumours large enough to be detected months or years after adjuvant treatment has been administered to these breast cancer patients. Subsequently,20% of breast cancer survivors have recurrences with metastatic lesions within 3-5 years after treatment.

It is a scientific fact that breast cancer stem cells which may remain in the tumour site even when surgical margins are clear can survive after postsurgical adjuvant chemotherapy and ionizing-radiation which is delivered to the chest wall after a mastectomy for reducing the risk of recurrence in breast cancer patients with tumors of 5 cm in size or larger,or with more than four positive lymph nodes.

These surviving breast cancer stem cells which are protected from DNA damage by specifically resistant to induction of apoptosis or type I PCD mechanisms become more mutant and malignant,fast spreading and resistant to chemo/radiotherapy  than the original tumour cells causing relapse of the disease which is associated with rise of new tumours and metastases leading to an extremely dismal prognosis [8]. 

Thus,the design of a novel therapeutic approach targeted towards breast cancer stem cells (BCSCs) is desperately needed for the complete eradication of tumour growth postsurgically with adjuvant treatment.This scenario seems like a science fiction with the present therapeutic capabilities resembling a field with numerous weeds of which no matter how many the whacker cuts,they come back much stronger because the roots, which in our case are the breast cancer stems cells (BCSCs),remain unharmed .
Thus,we need to identify breast cancer stem cells and target them with nanodelivery systems for their eradication sparing normal cells.This may give an answer to a life or death question

.
For this reason,we can use a theranostic approach against chemo/radioresistant breast cancer stem cells (BCSCs) combining noninvasive targeted nanoimaging with laser-nanosurgery based on nanophotothermolysis or near infrared plasmonic photothermal therapy (PPTT) under a personalized cancer medicine or personalized oncology approach based on pharmacogenomics and molecular targeting combined with nanomedicine and specifically nanooncology,as follows:

I] TARGETED NANOIMAGING: 
Immunotargeted near-infrared (NIR) contrast agents ,such as non-toxic pegylated gold-nanoparticles with covalently conjugated anti-CD44 antibodies or FAbs may be used for tumour imaging where breast cancer stem cells (BCSCs) via binding to their molecular receptors can be visualized using optical modalities for the measurement of light scattered by the gold-nanoparticles including phase sensitive optical coherence tomography (PSOCT)[9] ,and photoacoustic-tomography (PAT) which integrates high ultrasound resolution with the high optical contrast due to strong surface plasmon resonance[10]. 
In addition to an enhanced scattering signal and tunable longitudinal plasmon absorption,the gold-nanoparticles can provide optical contrast via absorption or luminescence.Furthermore,targeted nanogold particles may improve contrast with structural imaging modalities,such as MRI and CT-scans.Also,these contrast agents may be targeted to biomarkers under a molecular imaging approach for the production of information on the metabolic activity of breast cancer stem cells (BCSCs) using PET-scans with FDG.

II] IMMUNOTARGETED RADIATION THERAPY ENHANCEMENT (IRTE):
These immunotargeted pegylated gold nanoparticles can be used as X-ray contrast agents and radiosensitizers because they release in cancer stem cells and adjacent tumour cells energies including short range and low energy electrons, photoelectrons or characteristic X-ray,Auger electrons and a radiation dose enhancement due to enhanced photoelectric photon absorption by high-Z materials at kilovoltage photon energies.
Thus,since high-Z materials absorb ionizing-radiation (IR) more than tissue,there is production of highly localized heating which at a microscale causes burns in breast cancer stem cells (BCSCs) which generates reactive oxygen species (ROS), mitochondrial toxicity,release of cytokine,necrosis and apoptosis or type I programmed cell death (PCD).

III] POSTSURGICAL ADJUVANT NANOSURGERY VIA PHOTOABLATION FOR TARGETED PHOTOTHERMAL CANCER  THERAPY CAUSING NANOPHOTOTHERMOLYTIC  CANCER STEM CELL DEATH VIA PULSED NIR LASER:
Nanosurgery with NIR laser-pulses exerts adjuvant ablation of basal-like/triple(-) breast Ca stem-cells (BCSCs) by selective nano-photothermolysis mediated by pegylated-antiCD44 plasmonic gold-nanobombs.This non-invasive approach may be targeted to a specific molecular signature of  breast cancer stem cells (BCSCs) which have the potentiality to exert resistance to conventional anticancer treatments leading to metastasis.

Our immunotargeted NIR pegylated nanogold particles which are covalently conjugated to anti-CD44 Abs are used for highly specific molecular targeting of breast cancer stem cells (BCSCs).Our aim is to design a novel postsurgical adjuvant treatment facilitated by plasmonic laser nanosurgery (PLN) which will be targeted towards breast cancer stem cells (BCSCs) for the complete eradication of basal-like/triple(-) tumour growth.After preparing the plasmonic pegylated gold immunonanoparticles which are conjugated covalently with anti-CD44 Abs for selective molecular targeting of breast cancer stem cells (BCSCs),we administer them IV postsurgically for reaching the remaining BCSCs which overexpress CD44, circumventing with their stealth effect biological milieu interactions such as opsonin adsorption and subsequent reticuloendothelial system (RES) elimination, via the enhanced-permeability and retention (EPR) effect where bioconjugated nanoparticles passively accumulate at remaining   postsurgical tumour sites,which are characterized by leaky and immature vasculature that have wide fenestrations than normal mature blood vessels.

The nanoparticle size is 40-50 nm which is optimal for cellular entry of nanogold that has 600 times more absorption in cancer cells than normal cells.The particle size of nanogold-antiCD44 conjugate which is 40-50 nm is small enough to pass via the blood vessels of the remaining postsurgical tumor with fenestrae of 100 nm in diameter,and large enough to pass via the blood vessels of health organs which have fenestrae of no more than 5 nm in diameter[11].  

The anti-CD44 antibodies which are conjugated onto nanogolds target and bind specifically onto the cell surface transmembrane glycoprotein CD44 which is a hyaluronan receptor of breast cancer stem cells (BCSCs) activating cytoskeleton proteins such as microtubules (MTs) and actins which induce endocytotic internalization of nanoparticles that are transported into the cytoplasm of BCSCs by early and late endosomes.

Then,we irradiate with short NIR laser pulse of wavelength 1064 nm at 40 mJ/cm2 which is far below the safety standard for use of medical lasers for healthy cells and tissue which is 100 mJ/cm2.The short laser pulses exerted selective nanophotothermolysis only to the BCSCs which had nanogold particles intracellularly that caused necrotic and apoptotic or type I programmed cell death after there was thermal expansion of the gold nanoparticles which are characterized by a high plasmon resonance (SPR) absorption efficiency generating photoacoustic waves.The laser energy induced inside the BCSCs temperatures which have reached up to 950 C exerting hyperthermic effects which due to the short exposure will prevent extensive high temporative dissipation circumventing healthy cells.

BCSCs are very vulnerable to hyperthermia due to their rapid metabolic rates disrupting the signaling metabolic pathways,inducing acidosis and apoptosis due to the release of immunostimulants,such as heat-shock proteins [12].Also,the increases in temperature denaturate cytoskeletal and nuclear proteins inhibiting their synthesis by impending RNA and DNA polymerization.Furthermore, hyperthermia exerts cell membrane damage causing blebbing . Generally,thermal protein denaturation is caused in all intracellular proteins that are adjacent to the gold nanoparticles.The hyperthermic effects might damage the vasculative supply of the tumour cells,and cause disruption of homeostasis leading to microthrombosis.

Another mechanism consists of bubble formation around the gold-nanoparticles due to the explosive liquid evaporation (ELE) which is accompanied by acoustic and shock waves.This causes melting of the gold-nanoparticles which enhances tremendously their radius leading to their evaporation that forms small particles and gold vapor bubbles.

The next observed nonlinear mechanisms consisted of optical-breakdown with subsequent formation of plasma cavity,and generation of strong shock waves which cause explosion of the nanoparticles that act as nanobombs leading to their fragmentation and exerting extensive cellular damage via disintegration of organelles,and nuclear fragmentation which causes an apoptotic bystander killing effect.

Thus,the laser nanosurgery may lead to apoptosis of BCSCs via coagulation,and disruption which is caused by nanophotothermolysis that is associated with thermochemical transformation of vital cellular proteins,and explosive vaporization in the intracellular regions which are located near the gold nanobombs generating shock waves which are associated with supersonic expansion of dense vapor intracellularly which produces optical-plasma,and strong sound waves leading to photothermal apoptotic cell death (PACD) caused by oxidative stress and depolarization of the membranes of mitochondria which activate apoptotic caspases leading to fragmentation of the DNA.

This nanophotothermolysis of DNA caused by the thermal explosion of gold nanobombs due to possible Coulomb explosion via ionization of multiphotons and thermal explosion through electron photon excitation relaxation (EPER) eradicated the targeted BCSCs sparing the healthy adjacent breast cells.
Thus,we have the medical potentiality with targeted molecular imaging to identify the BCSCs after surgery,and subsequently as adjuvant eradicating treatment with nanosurgical laser ablation facilitated by a flexible optical fiber to selectively target the remaining BCSCs which cause metastasis under a personalized cancer medicine approach based on pharmacogenomics,and mediated by nanomedicine leading to their apoptotic cell death with a bystander killing effect by nanophotothermolysis without harming the adjacent healthy breast cells under a Trojan horse nanodelivery system which facilitates a “bow and arrow” theranostic approach circumventing the “one size fits all” approach.                          


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