LITERATURE REVIEW 1.1 STEM CELLS AND CANCER STEM CELLS
Stem cells
Stem cells (SCs) are unspecialized cells capable of developing into specialized cells, including blood cells The concept of "stem cell" was introduced by Russian histologist Alexander Maksimov in 1908 during the Congress of Hematologic Society in Berlin, where he proposed the existence of hematopoietic stem cells (HSCs).
Stem cells (SCs), particularly hematopoietic stem cells (HSCs) from bone marrow, play crucial roles in the regeneration of the human body While HSCs cannot transport oxygen in their undifferentiated state, they can become red blood cells capable of oxygen delivery Each day, millions of cells, including blood and skin cells, die and are replaced by new cells that differentiate from SCs To date, SCs have been identified and isolated in nearly every tissue of the human body, including bone marrow, adipose tissue, and peripheral blood.
Umbilical cord blood, along with components such as umbilical cords, umbilical cord membranes, and umbilical cord veins, plays a crucial role in medical research and treatment Wharton's jelly from the umbilical cord and the placenta, including the decidua basalis, are valuable for their regenerative properties Additionally, amniotic fluid and membranes, along with dental pulp, chorionic villi from the human placenta, fetal membranes, menstrual blood, and breast milk, are significant sources of stem cells and other biological materials that contribute to advancements in healthcare and regenerative medicine.
Stem cells (SCs) possess two key characteristics: the ability to self-renew, allowing them to undergo multiple cycles of cell division while remaining in an undifferentiated state This self-renewal is maintained through two distinct mechanisms.
Stem cell (SC) populations exhibit two types of division: asymmetric and symmetric In asymmetric division, one SC produces a father cell that remains identical to the original SC and a differentiated daughter cell Conversely, symmetric division results in either two differentiated daughter cells or two SCs that are identical to the original Additionally, stem cells possess potency, which refers to their ability to differentiate into various specialized cell types.
Figure 1 1 SC division and differentiation
Pluripotent embryonic stem cells (SCs), derived from the inner mass cells of a blastocyst, possess the unique ability to differentiate into any tissue in the body, except for the placenta In contrast, the cells of the morula are totipotent, meaning they can develop into all tissue types as well as the placenta.
Cancer stem cells
1.1.2.1 Tumor contains cancer cells with SC properties
The connection between stem cells (SCs) and cancer has been theorized for over 150 years, beginning with Durante's 1874 proposal that cancer originates from a rare population of normal cells with stem cell properties The following year, Cohnheim suggested that misplaced SCs during embryonic development could lead to tumor formation later in life This idea has driven extensive research into the existence of cancer stem cells (CSCs) In 1926, Bailey and Cushing further advanced the concept by asserting that cancer is initiated and sustained by a limited number of transformed precursor cells.
Research has confirmed that certain cancer cells, such as those derived from ascites fluid in rats and teratocarcinomas and leukemias in mice, can generate new tumors with heterogeneous phenotypes This hypothesis was notably articulated by Park et al (1971) and Hamburger and Salmon.
(1977) showed that some cancers contain a small cell population with properties of normal SCs, particularly myeloma cancer [129];[244]
Figure 1 2 Cell surface markers of CSCs in some varieties of cancers
(According to Morrison et al 2011 [223])
In a pivotal study by Lapidot et al (1994), cancer cells derived from human acute myeloid leukemia (AML) that expressed hematopoietic stem cell (HSC) markers were transplanted into NOD/SCID mice, successfully initiating leukemia In contrast, AML cells lacking HSC markers failed to induce cancer in these mice, establishing this transplantation method as a standard for identifying cancer stem cells (CSCs) Researchers have since focused on distinguishing CSCs through specific surface markers, enabling the isolation of these cells using monoclonal antibody-based sorting techniques The initial identification of a CSC marker was found in leukemia, characterized by the CD34 + CD38 - phenotype.
Bonnet and Dick showed that all leukaemia contains leukaemia CSCs with range 0.1-1% of the total cell population
Using the same technique, CSCs were demonstrated the existence in many others tumor types including brain, breast, colon, pancreas, prostate, lung, liver, skin and head and neck cancer [34];[79];[84];[96];[167];[187];[260];[285] (Fig
Tumors or cancers arise from malignant transformations due to mutations or genetic instability The Cancer Stem Cell (CSC) hypothesis posits that both stem cells and differentiated cells that acquire self-renewal capabilities can accumulate genetic alterations, allowing them to escape the regulatory control of their microenvironment and lead to cancer development This model suggests that CSCs can originate from both stem cells and differentiated cells, although it is challenging for differentiated cells to undergo sufficient genetic changes to gain self-renewal abilities and lose microenvironmental control Ultimately, the CSC model indicates that tumor progression, metastasis, and cancer recurrence are primarily driven by these cancer stem cells.
The Cancer Stem Cell (CSC) hypothesis in histopathology elucidates how alterations in tissue histochemistry correspond to the malignancy levels of tumors CSCs exhibit two key characteristics akin to stem cells: self-renewal, which facilitates significant evasion, and the potential for differentiation, leading to pronounced tumor phenotypic heterogeneity.
Figure 1 3 CSCs and tumor progression
(According to Dean et al 2005 [85])
Adult stem cells (SCs) typically proliferate more slowly than their differentiated progeny, contributing to their longevity However, this slower proliferation exposes them to more damaging agents over time, leading to the accumulation of mutations These mutations can be passed on to rapidly proliferating progeny, known as cancer stem cells (CSCs) Consequently, the progeny of normal adult SCs may exhibit characteristics of CSCs Research indicates that CSCs share several signaling pathways and markers with normal SCs, such as the Notch signaling pathway, which is prominently expressed in both breast CSCs and mammary SCs.
Cancer stem cells (CSCs) are characterized by their remarkable ability to initiate tumors from a small number of cells, leading to a high risk of recurrence This recurrence is primarily due to their self-renewal capabilities and strong resistance to both radiotherapy and chemotherapy Additionally, CSCs possess stem cell properties that allow them to differentiate into various cell types, contributing to significant cellular heterogeneity In contrast, mature cells do not have the ability to self-renew and exhibit lower proliferation potential.
Mature cells and stem cells (SCs) have the ability to transform into cancer stem cells (CSCs) through a process known as dedifferentiation, where mature cells regain properties characteristic of stem cells.
BREAST CANCER AND BREAST CANCER STEM CELLS
Breast cancer is the most prevalent cancer among women globally and a leading cause of cancer-related deaths, with over 1 million new cases and more than 410,000 fatalities each year The International Agency for Cancer Research reports that breast cancer represents 21% of all cancer cases in women worldwide In 1998, the incidence rate was 92.04 per 100,000 in Europe and 67.48 globally Even in developed nations, breast cancer accounted for 130,000 deaths in Europe and 40,000 in the US in 2004 The disease is increasingly common in developing countries, with Vietnam reporting the highest rates of breast cancer among women, showing an age-standardized rate of 20.3 per 100,000 in Hanoi (1998) and 16.0 per 100,000 in Ho Chi Minh City (2004) Breast cancer is recognized as the most or second most common cancer among women in Vietnam.
[4];[6];[18] Especially the age range (from 40-49) is accounted for 35.2% of cases [11]; 52.2% of cases [6], 47.8% of cases [4], 30.17% of cases [18] depending on countries
Breast cancer treatment currently involves surgery, cytotoxic drugs, hormonal therapies, and immunotherapy, achieving response rates of 60% to 80% for primary cancers and around 50% for metastatic cases Despite these treatments, 20% to 70% of patients experience relapse within five years, with recurrence often leading to increased therapy resistance and a higher mortality risk In Vietnam, 21.7% of patients treated with modified radical mastectomy of Scanlon face relapse within the same five-year period.
Over the past decade, Vietnam has made significant advancements in breast cancer research and treatment, revealing unique characteristics of the disease in the local population Notably, the combination of radiation therapy and cytotoxic therapy with Arimidex has resulted in an impressive four-year survival rate of 91.6%.
In metastatic breast cancer, pegylated liposomal doxorubicin has shown response rates between 27.27% for stable disease and 45.46% for progressive disease The TAC regimen (Taxotere, Doxorubicin, Cyclophosphamide) demonstrates a higher overall response rate of 70.1%, with a complete response rate of 51.1% and a partial response rate of 20.0% Notable genetic mutations and gene expression dysregulation have been identified in Vietnamese breast cancer, including over-expression of heparan sulfate interacting protein, p53 mutations linked to high histological grades, and mitochondrial D-loop mutations, while no BRCA1 and BRCA2 mutations were found in 95 patients studied Additionally, translational research has explored natural and semi-synthetic substances, such as curcumin and 3', 5, 7-triacetyl-4'-methoxyflavanon, for inducing apoptosis in MCF-7 cells Pathohistological studies indicate that higher histologic grades correlate with poorer breast cancer prognosis, and while ER and PR status do not vary by age, they are significantly associated with histological grades.
In 2003, Al-Hajj and colleagues identified cancer stem cells (CSCs) in human breast tumors, characterized by specific markers: CD44 + CD24 -/low ESA + and lin - (lacking CD2, CD3, CD10, CD16, CD18, CD31, CD64, and CD140b) Remarkably, as few as 200 of these CSCs were capable of forming tumors in NOD/SCID mice, while tens of thousands of other cells failed to do so The resulting tumors mirrored the phenotypic diversity of the original tumor and contained a minority of CD44 + CD24 -/dim lin - cells, which could be serially passaged to generate new tumors This CD44 + CD24 - phenotype has since been pivotal in identifying and isolating cancer cells with heightened tumorigenicity.
Figure 1 4 Results of Al-Hajj et al (2003) about BCSCs
A representative tumor was observed in a mouse at the CD44 + CD24 -/low Lin - injection site, while no tumor was present at the CD44 + CD24 + Lin - injection site Additionally, flow cytometry confirmed the presence of breast cancer stem cells (BCSCs) in human breast tumors.
In 2005, CD44 + CD24 -/dim breast cancer stem cells (BCSCs) were isolated from patient samples through in vitro cell culture, and in 2008, they were also derived from established breast cancer cell lines These BCSCs are capable of forming mammospheres during culture, which helps maintain mammary epithelial cells in an undifferentiated state The formation of mammospheres indicates that these cells are early progenitors or stem cells, possessing the potential to differentiate into all three mammary epithelial lineages.
Mammospheres derived from breast cancer cells are primarily composed of CD44 + CD24-/dim-or-low phenotype cells, which have the capability to induce tumors when injected into NOD/SCID mice Notably, only a small fraction of breast cancer stem cells (BCSCs) can generate secondary mammospheres Furthermore, cancer cell lines containing 90% BCSCs do not exhibit greater tumorigenicity compared to those with just 5%, suggesting that only a specific subgroup of BCSCs possesses self-renewing properties.
The CD44 + CD24 -/low phenotype of breast cancer stem cells (BCSCs) has led to the discovery of additional markers, notably the aldehyde dehydrogenase (ALDH) family of cytosolic isoenzymes, which play a crucial role in oxidizing intracellular aldehydes and facilitating early stem cell differentiation ALDH1, the predominant isoform in mammalian cells, exhibits high activity in hematopoietic stem cells (HSCs) and various cancer stem cells (CSCs) Research utilizing Aldefluor staining has successfully identified breast CSCs, revealing that ALDH-positive breast cancer cells demonstrate a significant tumorigenic potential in NOD/SCID mice, indicating that the ALDH + pool likely contains the CSC population The combination of CD44 + CD24 - and ALDH + phenotypes for selecting CSCs has shown that these cells possess higher tumorigenicity compared to those with only CD44 + CD24 - or ALDH + characteristics Recent strategies have emerged to enhance the identification and isolation efficiency of BCSCs.
Figure 1 5 SP profile for a fine needle aspirate taken from a male breast cancer patient
In a study, it was found that the side population (SP) constituted 0.6% of the total cell population when incubated with 2.5 µg/mL Hoechst 33342 dye Furthermore, the efflux of Hoechst dye from the SP population was partially inhibited by pre-treatment with a combination of 10 µM FTC and 50 µM verapamil before the Hoechst incubation.
Recently a different approach was reported by Sajithlal and collaborators
In a study involving MCF-7 cells, researchers labeled the cancer stem cell (CSC) population with GFP using the Oct3/4 promoter They found that only 1% of the cells expressed GFP, with most of these cells being CD44+ CD24- Notably, the GFP+ cells exhibited a tumorigenic potential 100-300 times greater than their counterparts and showed resistance to cytotoxic drugs.
In general, BCSCs can be identified using several methods [31];
- Flow cytometry—analysis of BCSC markers including CD44 [33], CD24
[290], CD49f [256] and ALDH1 [113] Cells are then sorted and characterised analyzed by clonogenicity, proliferation, differentiation and in vivo tumorigenicity assays
- Label retention studies—For example radioactive thymidine and BrdU [328];
- Quiescence—Pece and colleagues (2010) have isolated normal human mammary stem cells using the lipophilic dye, PKH26, which is retained by quiescent cells [249];
Functional assays reveal that SP cells exhibit a heightened capacity to efflux Hoechst 33342 dye, while ALDH-positive cells are detected through the Aldefluor assay, which identifies cells with elevated ALDH activity.
1.2.2.2 Important characteristics of BCSCs a Chemoresistance
Chemotherapy significantly enhances mammosphere formation in tumor cells, with a reported 14-fold increase in patients treated with these agents Mouse model studies indicate that chemotherapeutic exposure exerts selective pressure and inhibits the differentiation of cancer stem cells (CSCs), thereby elevating their proportion within tumors Research by Yu and colleagues demonstrated that tumors in mice treated with epirubicin were notably enriched in CD44 + CD24 - lin - cells, which exhibited a capacity to form mammospheres 20 times more than those derived from parental cell lines.
In another study, Shafee et al (2008) showed that the proportion of
Cisplatin treatment leads to a significant increase in CD29 hi CD24 med tumorigenic cells, with a fourfold rise compared to untreated primary tumors This chemoresistance in breast cancer stem cells (BCSCs) is partly attributed to the expression of ABC transporters A specific subpopulation of breast cancer cells, known as the "side population," demonstrates the ability to extrude the dye Hoechst 33342, indicating heightened ABC transporter activity This subpopulation shows a remarkable 30-fold increase in ABCG2 mRNA expression when compared to unsorted cells.
The loss of ABCG2 expression and drug efflux capacity during the differentiation of cancer stem cells (CSCs) into cancer cells may elucidate the primary chemotherapy resistance observed in breast cancer stem cells (BCSCs) Additionally, this phenomenon contributes to the radiation resistance seen in these cells.
BREAST CANCER STEM CELLS TARGETING THERAPY
1.3.1 Targeting on stemness of BCSCs
1.3.1.1 Directly targeting on BCSC self-renewal
Therapies targeting the stemness of breast cancer stem cells (BCSCs) aim to differentiate these cells into non-BCSCs, effectively eliminating their self-renewal capacity Once BCSCs lose this ability, they also lose their resistance to radiotherapy and chemotherapy, leading to reduced invasion In contrast to traditional cancer therapies that only kill tumor cells, CSC-targeted therapies focus on completely attacking BCSCs, potentially leading to tumor degeneration and preventing regrowth.
Chemo- and radioresistance significantly reduce the effectiveness of cancer treatments by activating anti-apoptotic signaling pathways that inhibit cell death Recent research has identified several molecular mechanisms that contribute to the resistance of breast cancer stem cells (BCSCs) against cytotoxic agents.
Figure 1 7 Differences in CSCs targeting therapy and traditional cancer therapy in breast cancer treatment
Breast cancer stem cells (BCSCs) exhibit over-expression of proteins associated with multidrug resistance and demonstrate elevated levels of anti-apoptotic proteins like survivin and BCL-XL Additionally, BCSCs possess enhanced DNA repair capabilities, contributing to their resistance against conventional therapies.
Figure 1 8 Targeting signal transduction pathways in BCSCs
This article presents a schematic illustration of critical signal transduction pathways and therapeutic targets, highlighting key agents in red The pathways discussed include Notch, Hedgehog, Wnt, and the HER2-Akt pathway, along with cytokine loops involving interleukins IL-6 and IL-8.
Self-renewal pathways are crucial for both stem cells (SCs) and cancer stem cells (CSCs), with key signaling pathways such as Wnt, Notch, and Hedgehog being prominently expressed in SCs These pathways play a significant role in maintaining the phenotype of breast cancer stem cells (BCSCs) during tumor growth Research has demonstrated that dysregulation of these pathways in the mammary gland can lead to breast cancer in mice Furthermore, dysregulation of these pathways is also observed in nearly all human BCSC lines.
The Her2-signaling pathway is crucial for self-renewal processes, and numerous therapies have been developed to target Her2, primarily aimed at treating Her2-overexpressing breast cancer Notable agents like trastuzumab and lapatinib have shown promise; in vivo studies indicate that these treatments can enhance progression-free and overall survival in patients with advanced disease Additionally, in vitro research demonstrates that trastuzumab can effectively reduce the population of breast cancer stem cells (BCSCs).
[174] However, these agents also met some limitations, especially nearly 50% of patients who respond to HER2-targeted agents relapse within a year [228]
1.3.1.2 Indirectly targeting on BCSC microenvironment
The microenvironment surrounding stem cells, known as the stem cell niche, plays a crucial role in tumor biology This niche comprises various cellular components, including inflammatory cells, fibroblasts, endothelial cells, and mesenchymal stem cells, all of which contribute to the tumor's development and progression.
Interleukin IL-6 and IL-8 have been identified as key regulators of breast cancer stem cell (CSC) self-renewal in both in vitro and xenograft models Research by Korkaya et al (2008) demonstrated that chemotherapy can elevate local levels of IL-8, subsequently increasing CSC populations Additionally, other studies have linked IL-6 and IL-8 to the development of metastasis and unfavorable patient outcomes.
Research indicates that cytokines like IL-6 and IL-8 are crucial in regulating cancer stem cells (CSCs) within their niche, highlighting the potential of micro-environment targeting therapies in cancer treatment For instance, the use of statins, known for their anti-inflammatory properties, has been associated with a reduced risk of breast cancer (Kochhar RKV, 2005) Additionally, antibodies that target the IL-8 receptor CXCR1, along with small molecules like repertaxin that inhibit CXCR1/CXCR2, have shown promise in suppressing tumor growth and metastasis.
1.3.2 Killing BCSCs by specific markers
1.3.2.1 Chemotherapy causes differentiation or apoptosis of BCSCs
Recent studies have focused on developing innovative treatments for breast cancer, including stealth liposomal daunorubicin combined with tamoxifen, which effectively eradicates both breast cancer cells and breast cancer stem cells (BCSCs) This formulation demonstrated a high concentration of active ingredients, with daunorubicin at 95% and tamoxifen at 90% Additionally, all-trans retinoic acid stealth liposomes paired with vinorelbine stealth liposomes have shown promising results in preventing tumor recurrence in mouse models Metformin, a common diabetes medication, has also been identified as an agent that selectively targets BCSCs and, when used alongside doxorubicin, significantly reduces tumor mass and prevents relapse The combination of differentiation agents like all-trans retinoic acid and metformin with anti-tumor drugs represents a potent strategy for eliminating CSCs and reducing the risk of cancer recurrence.
Dendritic cells (DCs) are among the most utilized immune cells in immunotherapy, particularly for breast cancer A novel approach involves using DCs loaded with killed breast cancer cell-derived antigens, which can capture these cells and, upon maturation, efficiently present MHC class I and II peptides to CD8+ and CD4+ T lymphocytes This process primes naive CD8+ T cells to differentiate into effector cytotoxic T lymphocytes (CTLs) Research by Saito et al identified CTLs specific to HLA-A2 restricted peptides from key breast tumor antigens, including cyclin B1, MUC-1, and survivin Additionally, some studies have fused tumor cells with DCs to enhance tumor antigen presentation, showing that DCs pulsed with apoptotic breast tumor cells can trigger effective antitumor T cell responses in vitro Animal models further indicate that vaccination with DC/tumor fusions can protect against tumor challenges and induce disease regression, with preclinical studies demonstrating that these fusion cells stimulate immune responses capable of lysing autologous tumor cells.
In a Phase I study involving 32 patients vaccinated with 10^5 to 4 x 10^6 fusion cells, there were no significant treatment-related toxicities or clinical signs of autoimmunity Notably, a subset of patients showed an increase in CD4+ and CD8+ T cells expressing intracellular IFN-gamma in response to tumor lysate exposure Among these patients, two experienced disease regressions, while five with renal carcinoma and one additional patient achieved disease stabilization.
In 1977, researchers identified transformed cell lines that were sensitized to human reovirus, an oncolytic virus known for its ability to selectively replicate in cancer cells without causing significant disease in humans.
Recent experiments have demonstrated that reovirus can effectively replicate in various human cancer cell lines, including those from the brain, breast, lymphoma, ovarian, bladder, spinal, and colon cancers As a result, reovirus has emerged as a potential anti-cancer therapeutic agent Intratumoral injections of reovirus have shown to induce tumor regression in immune-compromised mice with human tumors Furthermore, numerous promising anti-cancer agents based on oncolytic viruses are currently undergoing clinical trials, showing encouraging safety and efficacy results.
Recent studies indicate that reovirus can effectively infect various breast cancer cell lines, achieving over 50% cytolysis by day 7 post-infection Oncolytic reovirus shows promise in inducing tumor regression in breast cancer patients, with findings revealing that both breast cancer stem cells (BCSC) and non-BCSC populations respond similarly to reovirus treatment Although there are currently no clinical trials utilizing reovirus therapy, it holds significant potential as a future treatment option for breast cancer.
KNOCK DOWN GENE THERAPY AND IMMUNOTHERAPY
1.4.1 Knock down gene therapy for cancer
RNA interference (RNAi) is a post-transcriptional gene silencing technique that utilizes double-stranded RNA to downregulate specific gene expression There are two primary methods for delivering small interfering RNA (siRNA) to target cells: non-viral and viral vectors Viral vectors demonstrate higher delivery efficacy compared to non-viral methods, which involve the direct transfection of siRNA into cells In contrast, viral vectors introduce a sequence that is processed into short hairpin RNA (shRNA), ultimately yielding 21 bp siRNA after cleavage by the Dicer enzyme.
Figure 1 9 Some gene knock-down strategies
The use of siRNA for gene knockdown employs various strategies, including the inhibition of over-expressed oncogenes, promotion of apoptosis, regulation of the cell cycle, anti-angiogenesis, and enhancement of chemotherapy and radiotherapy efficacy Since its discovery, RNAi technology has emerged as a powerful approach for cancer gene therapy Initially recognized in transgenic plants, RNAi was first identified in the genome research of the worm Caenorhabditis elegans by Fire et al in 1998, while Elbashir et al later demonstrated the RNAi phenomenon in cultured mammalian cells in 2001.
Chemically synthesized siRNA can activate the interferon system in mammalian cells, resulting in protein synthesis shutdown and non-specific mRNA degradation To enhance efficacy, Kim et al developed siRNA in vitro using T7 RNA polymerase without the 5′-triphosphate to prevent the interferon response However, high doses of siRNA can still trigger interferon activation To address these issues, alternative viral vectors expressing shRNAs via RNA polymerase-III promoters are utilized, with the shRNA subsequently processed into approximately 21 bp siRNA by the Dicer enzyme from the RNAse III family.
Figure 1 10 Diagrams of three general ways of encoding siRNA in a plasmid or viral vector [70]
1.4.1.2 Non-viral vector vs viral vector
Non-viral vector RNA interference (RNAi) typically utilizes chemically synthesized components delivered into target cells via non-viral vehicles like lipophilic molecules, polymer vectors, and nanoparticles Research by Elbashir et al (2001) demonstrated the effectiveness of liposomes for siRNA delivery into cultured mammalian cells, while Bologna et al (2003) employed polyethylenimine (PEI) to transfect various nucleic acids, including siRNAs Additionally, Schiffelers et al (2004) utilized sterically stabilized nanoparticles for ligand-targeted delivery of siRNA oligonucleotides into tumor cells Although non-viral vectors are generally safer and easier to produce than their viral counterparts, their clinical application is hindered by low transfer efficiency Recently, viral vectors, such as the Mouse Moloney leukemia virus, have emerged as effective siRNA delivery systems, demonstrating high efficacy in targeting neoplastic cells across a broad range of hosts.
Abbas-Terki et al (2002) developed lentiviral vectors to deliver siRNA targeting enhanced green fluorescent protein (EGFP), resulting in silencing effects observed as early as 72 hours post-infection and lasting for at least 25 days Additionally, adenoviruses serve as a prominent alternative system, offering advantages over other viral vectors by efficiently introducing genetic material into host cells Notably, adenoviruses can be produced at high titers (up to 10^12 Pfu/mL) and can accommodate large gene fragments, enhancing their utility in genetic research and therapeutic applications.
Adenoviral vectors, such as Ad-p53, are capable of infecting a wide range of mammalian cell types and are relatively easy to handle, making them a popular choice in gene therapy However, their effectiveness can be limited, as demonstrated in a study involving 25 patients with non-small-cell lung cancer (NSCLC), where only 8% exhibited partial responses to the treatment This limited success may be attributed to the body's tolerance to repeated intra-tumoral injections of Ad-p53 and the challenges in achieving a 100% infection rate.
Figure 1 11 siRNA and shRNA activity [127]
1.4.1.3 siRNA strategies in cancer treatment a Silencing over-expressed oncogenes
Overexpression of oncogenes such as ras and myc is crucial in the development of cancer, with oncogenic mutations in the ras gene found in about 30% of all cancer cases Targeting these genes through silencing techniques has shown promising potential for cancer treatment Brummelkamp et al (2002) developed K-ras siRNAs delivered via viral vectors, resulting in a significant decrease in K-ras expression and tumor inhibition Similarly, Fleming et al (2005) demonstrated that silencing mutant K-ras with siRNA effectively inhibited pancreatic adenocarcinoma, highlighting the therapeutic potential of this approach in oncology.
K-ras and resulted in reducing malignant tumors formation, migration and angiogenesis in mice charged with tumor cells [108]
The c-Myc protein is significantly over-expressed in numerous human cancers, including 80% of breast cancers, 70% of colon cancers, 90% of gynecological cancers, and 50% of hepatocellular carcinomas Research indicates that silencing the c-Myc gene using siRNA leads to a reduction in tumor growth in nude mice, highlighting its potential role in promoting apoptosis.
Apoptosis, or programmed cell death, is crucial in cancer development and progression, as it depends on the balance between pro-apoptotic and anti-apoptotic proteins The Bcl-2 family proteins, located in mitochondria, are key anti-apoptotic regulators Inhibitors of apoptosis, such as NAIP, c-IAP1, c-IAP2, XIAP, Survivin, and BRUCE, also play significant roles in preventing cell death Targeting and knocking down these anti-apoptotic proteins can promote apoptosis in cancer cells For instance, the knockdown of XIAP, Survivin, and Bcl-2 in pancreatic cancer cell lines has been shown to induce apoptosis Similarly, inhibiting Survivin has been found to reduce cell proliferation and trigger apoptosis in human endometrial cancer.
Dysregulation of the cell cycle is a key factor in carcinogenesis, with specific regulatory proteins like cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs) playing crucial roles Cyclin E, in particular, is known to regulate the G1/S phase transition, and its overexpression can lead to G1 progression, resulting in genome instability and cancer development Targeting regulatory proteins, especially cyclin E through knockdown strategies, shows promise as a therapy to inhibit cancer cell growth Research by Li et al (2003) demonstrated that siRNA oligonucleotides can induce apoptosis and suppress tumor formation in hepatocarcinoma models in nude mice.
Research by He et al (2009) demonstrated that inhibiting cyclin E expression in breast cancer cells can effectively halt their cell cycle at the G1 phase, leading to reduced growth, differentiation, and proliferation, while also enhancing sensitivity to chemotherapy Similarly, silencing cyclin D1 using siRNA in MCF-7 cells resulted in decreased cell proliferation, G1 phase cell cycle arrest, and a loss of colony-forming ability These findings highlight the potential of targeting cyclin proteins to inhibit angiogenesis in breast cancer treatment.
Angiogenesis is essential for tumor growth and metastasis, with over 20 angiogenic factors identified, such as VEGF, TGF, bFGF, Angiogenin, and PDGF, alongside negative regulators like Angiostatin, Endostatin, Vasostatin, Tumstatin, and IL-12 Utilizing RNA interference (RNAi) to inhibit or reduce these angiogenic stimulators presents a promising anti-angiogenic strategy in cancer therapy, as noted by Chen et al.
Inhibiting VEGF-C expression through siRNA has been shown to reduce the growth of lymph node and lung metastasis (2005) Additionally, Shen et al (2007) demonstrated that knocking down vascular endothelial growth factor receptor 1 (VEGF-R1) using siRNA revealed its therapeutic potential in antitumor treatments.
1.4.2 Immunotherapy for cancer by dendritic cells
Immunotherapy for cancer is a treatment that enhances the immune system's ability to fight cancer using various agents, including vaccines, immune cell infusions, and cytokines This therapy works through several mechanisms: boosting the anti-tumor response by increasing effector cells or producing soluble mediators; reducing suppressor mechanisms; modifying tumor cells to enhance their immunogenicity; and improving tolerance to chemotherapy or radiotherapy, such as by stimulating bone marrow function with GM-CSF Key strategies in cancer immunotherapy include vaccination, increasing immune system activity, and addressing disease burden.
Cancer immunotherapy specifically targets and harnesses the patient's immune system to combat malignant tumor cells This innovative treatment can involve the injection of cancer vaccines, like Dendreon's Provenge, which trains the immune system to recognize and attack tumor cells Additionally, antibodies can be administered to stimulate the immune response against these cancerous cells Another promising approach is cell-based immunotherapy, where immune cells such as NK cells, LAK, CTLs, and DCs are either activated in vivo or in vitro and then reinfused into the patient While this method can yield significant results by enabling the immune system to effectively identify and destroy cancer cells, challenges remain, including the presence of unusual antigens on tumor cells and the rarity or absence of certain cell surface receptors.
MATERIALS - METHODS 2.1 MATERIALS
Instruments
Balance AZ214 Sartorius (Goettingen, Germany)
Cell CO 2 incubator CB150 Binder (Tuttlingen, Germany)
Centrifuge, big Allegre X15R Beckman Coulter, USA
Centrifuge, small 5810 Eppendorf (Humburg, Germnany) Clean bench, class II BioUltra IIA Telstar (Terrassa, Spain)
Cryostat CM1850-UV Leica (Germany)
FACS Sorter FACSJazz BD Biosciences (Franklin, USA) FACS analyzer FACSCalibur BD Biosciences (Franklin, USA)
Gel capture Gel-docIt UVP (Upland, CA)
Gel Electrophoresis Sub-CellGT Cell Biorad (Hercules, USA)
Gene sequencer and gene analyzer
GenomeLab Beckman Coulter (Brea, USA)
Fluoresence microscope Axio Observer Carl-Zeiss (Oberkochen,
Germany) Inverted microscope AxioVert 40C Carl-Zeiss (Oberkochen,
Germany) Magnetic stirrer CB300 Stuart (Staffordshire, UK)
Megnetic cell sorter MultiStand Miltenyi (Germany)
Orbital shaker SSM1 Stuart (Staffordshire, UK)
Osmonmetter Osmomat 030 Gonotec (Berlin, Germany)
PCR cycler Gradient pro S Eppendorf (Humburg, Germnany) PCR realtime cycler Realplex Pro S Eppendorf (Humburg, Germnany) Photometer Bio Photometer Eppendorf (Humburg, Germnany)
Plate reader DTX880 Beckman Coulter (Brea, USA)
Vortexer SA8 Stuart (Staffordshire, UK)
Water bath WNE10 Memmert (Germany)
Chemicals and Consumables
Agarose Sigma-Aldrich (St Louis, MO, USA)
Cell culture flask T-25 Nunc (Denmark)
Cell culture dish 35 mm Nunc (Denmark)
6 well dish culture Corning (Tewksbury, MA, USA)
12 well dish culture Corning (Tewksbury, MA, USA)
24 well dish culture Corning (Tewksbury, MA, USA)
96 well dish culture Corning (Tewksbury, MA, USA)
Glucose Sigma-Aldrich (St Louis, MO, USA)
Microscope slides Langenbrinck (Teningen, Germany)
Paraformadehyde Santa Cruz Biotechnology Inc CA
Sucrose Sigma-Aldrich (St Louis, MO, USA)
Tris Sigma-Aldrich (St Louis, MO, USA)
Triton X-100 Sigma-Aldrich (St Louis, MO, USA)
Trypsin Sigma-Aldrich (St Louis, MO, USA)
Polybrene Sigma-Aldrich (St Louis, MO, USA)
Puromycine dihydrochloride Sigma-Aldrich (St Louis, MO, USA)
General consumables and chemicals were purchased from Sigma-Aldrich (St Louis, MO)
Solutions, cell culture medium, growth factors and antibodies
Solutions and cell culture medium Company
FBS Gibco (Invitrogen, Carlsbad, USA)
Ultra water GeneWorld (HCM, VN)
DMEM/F12 Sigma-Aldrich (St Louis, MO, USA)
MEGS Gibco (Invitrogen, Carlsbad, USA)
EGF Sigma-Aldrich (St Louis, MO, USA)
FGF-2 Sigma-Aldrich (St Louis, MO, USA)
Insulin Sigma-Aldrich (St Louis, MO, USA)
RPMI 1640 Sigma-Aldrich (St Louis, MO, USA)
L-glutamin 100 mM Sigma-Aldrich (St Louis, MO, USA)
BSA Sigma-Aldrich (St Louis, MO, USA)
CD44 shRNA vector Santa Cruz Biotechnology Inc., Canada CD44 siRNA vector Santa Cruz Biotechnology Inc., Canada
Verapamil Sigma-Aldrich (St Louis, MO, USA)
Doxorubicin Sigma-Aldrich (St Louis, MO, USA)
GFP lentiviral vector Santa Cruz Biotechnology Inc., Canada Antibiotic-mycotic 100X Sigma-Aldrich (St Louis, MO, USA)
HEPES 1M Sigma-Aldrich (St Louis, MO, USA)
Trypsin/EDTA 0.25% Sigma-Aldrich (St Louis, MO, USA)
Fascflow solution BD Bioscience, Franklin, USA
IL-4 Santa Cruz Biotechnology Inc., Canada
GMCSF Santa Cruz Biotechnology Inc., Canada
TNF-alpha Santa Cruz Biotechnology Inc., Canada
2-mercaptoethanol Sigma-Aldrich (St Louis, MO, USA)
PHA Sigma-Aldrich (St Louis, MO, USA)
DMSO Sigma-Aldrich (St Louis, MO, USA)
FITC-dextran Sigma-Aldrich (St Louis, MO, USA)
Vybrant Dil CM Invitrogen, Carlsbad, USA
FITC Rat Anti-mouse CD40 BD Pharmingen, USA
FITC Rat Anti-mouse CD80 BD Pharmingen, USA
PE Rat Anti-mouse CD86 BD Pharmingen, USA
FITC Rat Anti-mouse CD14 BD Pharmingen, USA
FITC Mouse Anti-human CD24 BD Pharmingen, USA
PE Rat Anti-mouse CD8 BD Pharmingen, USA
PerCP Rat Anti-mouse CD45 BD Pharmingen, USA
PE Mouse Anti-human CD44 BD Pharmingen, USA
IgG Isotypes Santa Cruz Biotechnology, Inc, CA
Hoescht 33342 Sigma-Aldrich (St Louis, MO, USA)
Kits
Realtime PCR SYBR kits Sigma-Aldrich, St Louis, MO, USA
RNA Isolation Kit Fermentas, Maryland, USA
GenomeLab GeXP Start Kit Beckman Coulter, Brea, USA e-myco Mycoplasma PCR Detection kit Intron Biotechnology Inc., Korea
(Access RT-PCR system kit)
Promega, Madison, WI, USA siRNA Transfection Kit Santa Cruz Biotechnology Inc., Canada shRNA Lentiviral transfection Kit Santa Cruz Biotechnology Inc., Canada Annexin V and PI kit
Cell growth determination kit, MTT based
Sigma-Aldrich, St Louis, MO, USA
High Sensitivity IL12 Human ELISA
Pro-PREP Solution Intron Biotechnology Inc., Korea
Biological samples
- Human breast malignant tumors were obtained from Oncology Hospital at Ho Chi
In Minh City, Vietnam, all tumors were verified as malignant through standard assays conducted by the hospital The tumor samples were transferred to the laboratory in phosphate-buffered saline (PBS) enriched with 2X antibiotic-mycotic (Sigma-Aldrich, Louis St, MO) while maintaining a cool temperature.
- NOD/SCID mice, 5–6-week-old, NOD.CB17-Prkdcscid/J) (Charles River
Laboratories): were purchased from Jax Laboratory, USA All mice were hold in sterile room (Figure 2.2), supplied with food sterilied by gamma radiation; and water sterilied by autoclave
(A) Individually Ventilated Cage system, (B) An Individually Ventilated Cage (Techniplast, Louviers, France), (C) Biosafety Cabinet Class III used to manipulate mice (ESCO).
METHODS
This research was performed at Stem cell Research and Application Laboratory, University of Science, Vietnam National University, Ho Chi Minh city, Vietnam
Address: 227 Nguyen Van Cu, District 5, Ho Chi Minh city, Vietnam
This research was performed from May, 2009 to September, 2011
Breast cancer cells were isolated from tumor specimens obtained from consenting patients at the Oncology Hospital in Ho Chi Minh City, Vietnam, following the established protocol by Speirs et al (1998) The tumor biopsies were thoroughly washed with phosphate-buffered saline (PBS) containing antibiotic-mycotic and then dissected into small fragments of approximately 1-2 mm³ These fragments were re-suspended in an appropriate culture medium and placed into 35-mm culture dishes for incubation at 37°C with 5% CO2, with medium changes occurring every three days.
In our primary culture, we utilized two types of media Initially, we modified a medium based on Speirs et al (1998), originally intended for isolating epithelial cells from breast tumors, by enhancing it with 5% FBS to promote stromal cell growth This modified medium consisted of DMEM/F12 with 1X antibiotic-mycotic, 10 mM HEPES, 0.075% BSA, 10 ng/mL cholera toxin, 0.5 µg/mL hydrocortisone, 5 µg/mL insulin, and 5 ng/mL EGF In the second phase, we employed M171 medium supplemented with MEGS from Invitrogen, which is specifically designed for culturing mammary epithelial cells.
The sub-culture was carried out according to Araujo et al (1999) [35]
Cultured cells in a flask were treated with 0.25% trypsin/EDTA (Sigma-Aldrich, MO) until they rounded up and began to flake off Following this, the cell suspension was centrifuged to eliminate the trypsin, and the resulting pellets were re-suspended in an appropriate culture medium The dilution factor applied during sub-culture was 1:3.
BCSCs were cultured at a density of 1,000 cells/mL in serum-free DMEM/F12 medium, enriched with 10 ng/mL bFGF, 20 ng/mL EGF, 5 ng/mL insulin, and 0.4% BSA, following the methodology established by Ponti et al (2005).
Cells were cultured as non-adherent spherical clusters, commonly referred to as "spheres" or "mammospheres." After a 7-day culture period, the quantity of spheres in each well was assessed.
2.2.2 GFP transgenesis and establishment of GFP expressing cells
We established breast cancer cells and breast cancer stem cells (BCSCs) that stably express the GFP gene using a copGFP lentiviral vector The transduction was performed according to the manufacturer's instructions from Santa Cruz Biotechnology.
Transduced cells were cultured in a medium with 10 µg/ml puromycin dihydrochloride for one week to select for GFP-expressing cells The expression of GFP was validated using fluorescent microscopy and flow cytometry.
There were three methods used to sort interest cells in this research
The tube-catcher based cell sorting method was employed to isolate breast cancer cells by removing CD90 positive stromal cells Single-cell suspensions were prepared from cell populations at passages 2-4, with 10^7 cells re-suspended in a 200 µL PBS/EDTA solution and stained with 20 µL anti-CD90-PE After a 30-minute incubation in the dark at room temperature, the breast cancer cell population was identified as CD90 negative on the SSC-CD90-PE histogram This population was sorted using a tube-catcher based cell sorter on a FACSCalibur machine (BD Bioscience, Franklin Lakes, New Jersey), following the manufacturer's guidelines Due to initial low purification rates (95% were used for further research
2.2.4 Immunophenotype analysis by flow cytometry
Cells were washed twice in PBS with 1% BSA, followed by a 15-minute incubation on ice with IgG to block Fc receptors Subsequently, the cells were stained with anti-monoclonal antibodies at 4°C for 30 minutes After washing, analysis was conducted using a FACSCalibur flow cytometer and CellQuest Pro software, collecting data from 10,000 events.
The analysis of breast cancer cell populations focused on the expression of the CD24 and CD90 markers, with CD24 serving as a breast cancer marker and CD90 indicating stromal cells Cells were stained using 20 µl of CD24-FITC and CD90-PE antibodies from BD Bioscience Candidate breast cancer stem cells (BCSCs) were identified by their CD44+ CD24- phenotype, confirmed through staining with 20 µl of anti-CD44-PE and 20 µl of anti-CD24-FITC antibodies from BD Pharmingen.
To define the dendritic cells, they were stained with the following antibodies conjugated with FITC: anti-CD14, anti-CD40, anti-CD80, and anti-CD86 (BD Biosciences, Pharmingen)
Cells were fixed using 4% paraformaldehyde and washed with PBS before being incubated with a mouse anti-human CD24 monoclonal antibody, followed by a FITC-conjugated goat anti-mouse antibody Negative controls for the immunocytochemistry assays were performed by omitting the primary antibody Nuclei were stained with Hoechst 33342, and images were captured using a Carl-Zeiss microscope system equipped with a monochromatic camera.
2.2.6 Knock-down of CD44 on BCSCs
2.2.6.1 CD44 down regulation by siRNA
The CD44 small interfering RNA (siRNA) sequences utilized for knocking down various CD44 isoforms were 5′-AGC TCT GAG CAT CGG ATT T-3′, 5′-TGG CTG ATC ATC TTG GCA T-3′, and 5′-CAC CTC CCA GTA TGA CAC A-3′ These siRNAs were transiently transfected using a siRNA Transfection kit from Santa Cruz Biotechnology Inc., following the manufacturer's protocol Specifically, 1 µg of siRNA was combined with 1 mL of transfection medium and reagent, then incubated with 2 x 10^5 adherent cells for 5–7 hours at 37°C in a 5% CO2 environment, after which the medium was replaced.