Novel Strategies for Treating Breast Cancer: A Comprehensive Review

Introduction

The mammary gland functions as a highly dynamic organ that undergoes multiple stages of transformation. During each estrus cycle during pregnancy, the mammary epithelium proliferates and differentiates in response to both systemic and local cues.

A hierarchical array of progenitors and mammary stem cells (MaSCs), which normally maintain organ homeostasis, promote alveolar formation during pregnancy, and drive mammary ductal elongation and branching, orchestrates these changes [1]. The most frequent malignancy among women diagnosed globally is breast cancer (BC) [2]. Metastasis development is the primary driver of mortality for most cancer patients [3]. The most complicated and lethal event is metastasis. The interface between the tumor and the surrounding tissue is where tumor cells invade, either by singlecell or, less frequently but more effectively, collective migration [4]. The final and most deadly form of cancer is metastasis, which is the cancer cells’ growth in organs other than the one they started in [5]. The most recent statistics from the journal CA Cancer indicate that BC continues to be on the rise and has evolved into one of the most prevalent malignant tumors [6]. This type of cancer is a complicated condition impacted by both genetic and environmental causes [7]. It is important to note that it is the most frequent malignancy among women, accounting for over 316,000 instances of cancer in the US each year. Ductal carcinoma in situ (DCIS), or invasive ductal carcinoma in the milk ducts, is the initial stage of the majority of BCs. Mutational changes over time cause this cancer to develop, which occurs mostly in the ductal epithelium [7]. The World Health Organization (WHO) predicts that, by 2030, there will probably be a considerable rise in the incidence of this kind of cancer as a result of major changes in lifestyles [8]. It is worth noting that this cancer is usually classified using two perspectives: molecular and histopathological. BC in situ and invasive BC are two categories that are divided into different histopathological subtypes. Locally, there are two primary types of BC: lobular and ductal. There are five categories for ductal cancer: comedone, cribiform, micropapillary, papillary, and solid. Lobular classification is based on low pathogenicity. Invasive lobular cancer is classified as tubular ductal, mucinous medullary, infiltrative ductal, and invasive lobular. Molecularly, they are classified into the following groups: Luminal B (HR+[ER+ AND/OR PR+]HER2-/+) -Her2 positive (HR-[ER-PR-]HER2+) - Luminal A (HR+[ER+ AND/OR PR+]HER2-) - TNBC (HR-[ER- PR-]HER2-) – Normal like (HR+[ER+ AND/OR PR+]HER2-) [9-11]. Risk factors for this type of cancer include advanced age, female gender, having a positive family history, early menarche, thick breast tissue, hormone therapy, late menopause, not being pregnant or breastfeeding, and having chest radiation therapy in the past [12]. Cancer is the world’s second leading cause of mortality, after heart disease. One-third of women and half of men in the US will have cancer at some stage during their lifespan. Because of early detection and medical care, millions of people with cancer are now surviving longer [13]. With respect to the sort of malignancy and  cancer, there are a number of cancer therapies available, including surgery, hormone therapy, radiation, chemotherapy, immune targeting, stem cell transplantation, or a mix of these interventions [14].

Recently, significant progress has been made in many of the processes that lead to advances in BC treatment and how they are targeted (Figure 1). Combination techniques, such as multiple targeted therapies or “traditional” chemotherapy, including arsenic and platinum compounds, have a synergistic effect [15]. New treatment modalities, including biological molecules, pharmaceuticals, and immunomodulatory interventions, are used to decrease long-term survival periods for metastatic cancer while simultaneously raising rates of mortality, even in cases where the extraordinary therapy threshold has not been achieved [16]. Many in vitro and in vivo investigations regarding antioxidant therapies over the past few decades have shown that daily in take of a specific dosage of antioxidant nutrients is inversely associated with cancer risk while also improving the efficacy of treatment, despite the fact that randomized clinical trials have generated inconsistent findings that are believed to be an actual barrier for the widespread adoption of supplements with antioxidants in the treatment of cancer. These contradictory results can be caused by a variety of factors, such as dosage, synergy, bioavailability of used antioxidants, kind of cancer, patients’ health status, lifestyle, tendency to utilize supplements, and research duration. Thus, to provide a secure and effective treatment, more regulated and clearly defined clinical studies using more recent techniques should be conducted. Thus, in order to develop a safe and efficient antioxidant supplementation system for the cancer treatment, more carefully planned and specified clinical trials using more recent methodologies need to be conducted. In a similar vein, in vivo research ought to take precedence over comprehensive studies into novel antioxidant compounds from algae and their purification techniques. The specific antioxidants found in the different organic and aqueous extracts that are currently showing antioxidant and cancer fighting abilities in vitro, in addition to their effects on the cell system and potential to enhance the effects of chemotherapy drugs, need further investigation [17]. Physical examination, imaging, especially mammography, and tissue biopsy are essential for the diagnosis of this cancer [18]. We aim to investigate the possible uses of algae, CSCs, probiotics, and nanoparticles in the medical treatment of BC according to the data provided in this introduction.

Nanoparticles in Breast Cancer

Nanoparticles (NPs), which are microscopic particles of material measuring between 1-100nm in diameter, are used in nanotechnology [19]. The creation of nanomedicine agents through rapid progress in nanotechnology holds great promise for improving cancer therapy techniques [20] (Table 1). A new avenue in the treatment of cancer is presented by nanotechnology [21] (Fig. 2). These particles are highly modifiable for binding to cytoplasmic or nuclear receptor sites, the microenvironment, or the membranes of cancer cells [22]. In general, NPs can be classified as either inorganic or organic. Lipid and polymeric NPs are examples of organic NPs. Polymers comprise both homopolymers and copolymers. Homopolymer is categorized as a dendrimer, nanosphere, hydrogel nanocapsule, and nanocapsule. copolymer divided into polymerosomes and micelles. Lipid NPs comprised liposomes, solid lipid NPs, and micelles. Among the inorganic nanoparticles were carbon nanotube-ceramic NPs and metallic nanoparticle-fullerenes. Nanotechnology offers a more focused approach to addressing cancer-related challenges. Treating cancer disorders with a combination of nanocarriers and ligand targeting is a successful treatment approach. Cancer patients can benefit greatly from chemotherapy treatments, and using NPs for the treatment of cancer is additionally advantageous [23, 24]. Numerous NPs targeting malignant cells specifically while preserving healthy organs and tissues have been discovered recently. Consequently, the application of targeted drug delivery systems mediated by them in the treatment of BC has shown promise. Apart from their ability to target malignancies and reduce adverse reactions, NPs have a number of particular characteristics making them appealing for use in cancer treatment. These features include low toxicity, good flexibility, and ease of preparation [25]. In addition to medications, genes, and imaging agents that can identify tumors, recent research has demonstrated that NPs offer a number of benefits for the treatment and diagnosis of tumors [20]. Gold NPs are one type of NPs used in the treatment of BC. One form of nanomaterials employed to treat BC is gold nanoparticles (Au NPs). These NPs readily penetrate and stay in tumor vasculature because of their increased permeability and retention effect since the openings found in these channels, which can vary in diameter between 100 nm and 2 μm, are wider compared to the gaps that exist in the endothelium lining of healthy capillaries [26]. Alternatives that offer promise for the creation of novel anticancer medications are natural ingredients. Curcuma longa Linn, or Turmeric, is a plant whose rhizomes produce curcumin, also called diferuloylmethane, which is yellow in color. Curcuminoids and polyphenols are most abundant in the root of the plant. Due to its shown anti-inflammatory and antioxidant properties, curcumin is frequently utilized in culinary medicine and has been associated with conventional medicines. Kotan and associates originally revealed its anticancer effects in lymphoma models and animal cells in 1985. In both in vivo and in vitro models, curcumin has anti-metastatic, anti-invasive, and antiangiogenic capabilities in addition to inhibiting BC cell proliferation and inducing apoptotic cell death [27]. Curcumin has also demonstrated potential in reversing the treatment-resistant behavior of BC cells resistant to doxorubicin (MDA-MB-231/ DOX and MCF-7/DOX). Additionally, curcumin upregulated aldehyde dehydrogenase-1 and decreased the expression of the P-glycoprotein-mediated multidrug resistance gene, increasing the susceptibility of MCF-7 BC cells to paclitaxel. Regretfully, curcumin’s limited water solubility and poor bioavailability restrict its medicinal use. Therefore, in order to increase curcumin’s bioavailability and effectiveness, some research has looked into delivering it to BC cells using NPs [28-30]. Theranostics has a very promising future in the field of cancer therapy because of the effective advancement of dual-functionality NPs that are capable of monitoring/ imaging and treating a pharmaceutical disease. High multiplexibility makes it easier to combine tailored medication delivery at the right place to specifically kill tumor cells by conjugating ligands to NPs [31]. By reducing the pain associated with conventional chemotherapy, addressing current clinical obstacles, and determining the direction of future therapeutic possibilities, NPs are being meticulously selected to significantly advance the goals of BC therapy and diagnostics [32]. Combining medications with NPs can have positive anti-tumor effects. For instance, doxorubicin and mannose-modified DOX-nanoparticles (DOX-AS-M-NPs) remarkably decreased the overall number of tumors in mice and effectively controlled the growth of tumors [33]. The directed responses of triple stimuli of gold nanorods mixed with hyaluronic acid and glutathione during the PDT-PTT protocol targeting MCF-7 cell lines were established by Xu et al. in a prospective investigation. The compounds had a circulatory half-life of 1.9 hours and an accumulation rate of approximately 12.8% in tumor tissue. Innovative technologies provide real-time intervention tracking, optimized heat generation, reduced adverse reactions, and unprecedented accuracy in targeting malignant cells. As the profession advances, combination and customization treatments are becoming commonplace, opening the door for an era in which cancer treatments will become as unique as each patient [34].

Figure 2: Current organic and inorganic nanoparticles used in cancer therapy.

Table 1: Classification and applications of nanoparticles in cancer therapy.

Immunotherapy: A novel approach to treating breast cancer

In 1891, William B. Coley treated a patient with incurable cancer with an injection of streptococcal germs. He discovered that live or inactivated microorganisms could induce sarcoma remission, marking the inception of immunotherapy. Nevertheless, the relationship between immune cells and melanoma was not identified until the 1980s and 1990s, when researchers also developed the notion of cancer immunotherapy [39]. As an additional therapeutic option, cancer immunotherapy has advanced dramatically in recent years and rounds out the available treatment options. Unlike previous treatment approaches, immunotherapy’s main goals are to enhance the quality of life for those who are afflicted and stop the cancer from spreading metastatically [48]. Metastatic triple-negative breast cancer (TNBC), a kind of BC, is a disease that is aggressive, has a poor prognosis, and has few therapeutic choices. One intriguing new treatment option for this challenging condition is immunotherapy [49]. With the approval of autologous cellular immunotherapy, the field of cancer immunotherapy has lately experienced a major boost after many unsatisfactory attempts and clear clinical failures [50] (Table 2). Hematologic malignancies can now be effectively treated with cellular immunotherapy; however, outcomes in solid tumors have been less favorable due to their intrinsic heterogeneity and complex tumor microenvironment [51]. Adoptive cellular therapy (ACT), a different kind of immunotherapy that is quickly gaining popularity in clinical studies, entails separating T cells from a donor or patient, altering and/or growing them ex vivo, and then reintroducing them into the patient [52]. Adoptive cellular transfer treatment involves transferring specific HLA-matched allogeneic or autologous immune cells, which were successfully activated in vitro, to the patient for the purpose of triggering an anti-tumor immune response [51]. Innate lymphocytes known as natural killer (NK) cells are essential for eradicating cancerous cells and managing viral infections. The ability of NK cells to eliminate targeted cells without the need for prior sensitization was what first attracted their recognition [53]. Clinical efforts to apply adoptive NK cell transfer to solid tumors were motivated by the strategy’s early success in treating hematologic malignancies. In vitro NK cells exhibit cytotoxicity against a broad spectrum of tumor cells belonging to solid cancer types. Adoptively transferred NK cells have also been shown to exhibit antitumor effects in vivo in preclinical xenograft mouse models of metastatic colorectal cancer, glioma, and ovarian cancer [54]. The CAR-NK cell idea was first studied preclinically utilizing the NK92 cell line, which was taken from a 50-year-old male patient with extranodal NK cell lymphoma (ENKL). Because of its relatively easy in vitro multiplication, the NK-92 cell line promises a potentially limitless source of CARNK cells. NK-92 cells must be exposed to radiation in order to decrease their ability to engraft in vivo since they originate from a malignant lineage [55]. It’s important to remember that CAR-NK cells might be an “off-the-shelf” product, even though allogeneic CAR-T cell therapies are still being researched [56].

The research of BC immunology has advanced significantly during the last decade (Figure 3). Twenty years ago, this cancer was thought to be non-immunogenic, and immunotherapy was not intended to be used to treat it. Today, we know that most patients with this cancer have evidence of an adaptive immune response to their cancer, either tumor-specific or peripheral blood-based [57]. Because tumor cells might develop new defense mechanisms to evade and outlive the immune response, immunotherapy has become a popular treatment option to fill in the holes in the current paradigm for treating BC. Thus, a better understanding of immune bypassing of malignant cells and monoclonal antibodies against PD-1 and PD-L1 can help in the diagnosis of this malignancy [58]. The impressive results of cancer immunotherapy in treating acute lung cancer, lymphoblastic leukemia, melanoma, and other cancers show that T cells can act as an exogenous tumor suppressor. The development of effective immunotherapy for people with BC starts to show results once it is underway. When it comes to treating individuals with triplenegative disease and advanced BC, monoclonal antibodies PD-l1 or PD-1 (mAb) show the most promise. The objective response rate (ORR) in these investigations ranges from 12-19% [59]. At various stages of development and puberty, including postnatal puberty and pregnancy, the breast stroma contains different kinds of immune cells, making immunity essential for healthy breast growth [60]. Since certain monoclonal antibodies (mAbs), including trastuzumab, have long been employed as therapeutic medicines for treating tumors in this malignancy, passive immunotherapy does not constitute a novel approach to treating this disease. Certain alternative methods, such as immune checkpoint inhibitors (ICIs) and antibody drug conjugates (ADCs), have garnered more interest recently as a preventative and therapeutic approach for this malignancy [57]. Appropriate antigen delivery systems and a thorough comprehension of the immune signaling pathways controlling reactions throughout the tumor microenvironment are necessary for effective therapeutic treatments [59]. With the development of immune validation at TNBC and immunological checkpoint inhibition (ICI) in solid tumors, immunotherapy has become more popular. Researchers are making progress by focusing on several safety checkpoints on multiple treatment lines for TNBC based on the accumulation of clinical studies. These involve programmed death inhibitors-1 (PD-1), antigen inhibitor-4 associated with cytotoxic T lymphocytes (CTLA-4), and other innovative immunotherapy techniques (e.g., cancer-testicular antigen (CTA), individual peptide vaccine, vaccine RNA, and modified T cells containing chimeric antigen receptor (CAR-T)) [61]. Since its molecular characteristics were discovered, BC has undergone significant change. It is now associated with immune markers (such as lymphocyte tumor penetration and PD-L1), genomic markers (such as PIK3CA, BRCA2, and BRCA1), and histochemical markers (such as iodine, PR, HER2 [URBB2], and protein proliferation markers Ki-67 [MKI67]). Typical treatments for metastatic BC comprise targeted medications such as PARP inhibitors, PI3K inhibitors, CDK4 and CDK6 inhibitors, and anti-PD-L1 immunotherapy, based on the tumor type and molecular profile. The diversity of available treatments reflects the complexity of modern care for this cancer [62]. Novel approaches to treating TNBC may be brought about by developments in immunotherapy, which stimulate the immune system of the host to eliminate tumor cells. Current research in this area focuses on combining NK cell therapy, PARP inhibitors, chemotherapy, and cancer vaccines with ICI. Furthermore, plant diets high in plant chemicals can efficiently modulate signaling molecules related to the safety checkpoint, such as PD-1/PD-l1, which is why nutrients are frequently utilized in the TNBC treatment and prevention. Additionally, by specifically targeting cancer cells and controlling immune responses, for example, specially made NPs containing ligands on their surface can help deliver drugs more effectively. Furthermore, one effective strategy for overcoming multidrug resistance may be to use nanodrug delivery devices. However, a number of restrictions, such as inadequate therapeutic characteristics and poor oral biological access make their use complicated [63]. Although several novel treatment methods are currently in the clinical development stage, they are desperately needed for tackling the weakened anticancer immunity in TNBC. The IL-2 pathway agonist bempegaldesleukin, which specifically activates the IL-2b receptor to promote effector T cell growth while reducing proliferation of regulatory T cells, is one potential tactic. With an objective response rate (ORR) of 13.2%, the phase I/II PIVOT-02 study, which included 38 patients with metastatic TNBC who received bempegaldesleukin in addition to nivolumab, showed persistent responses regardless of PD-L1 expression rates. In TNBC, ongoing research is concentrating on more studies that combine bempegaldesleukin with chemotherapy and anti-PD-1/L1 treatments. The intratumoral toll-like receptor 9 (TLR9) agonist SD-101 is a different possible immunotherapeutic approach. It increases CD8+ T cell infiltration into malignancies by causing plasmacytoid dendritic cells (DCs) to release interferon-α and develop into antigen-presenting cells. As opposed to doxorubicin, paclitaxel, and cyclophosphamide alone, the I-SPY 2 study is now evaluating the possible pathological complete response (PCR) resulting from neoadjuvant SD-101 and pembrolizumab when combined with weekly paclitaxel following doxorubicin and cyclophosphamide [64-69].  According to experimental findings, 20% of TNBC patients have PD-L1, suggesting that this specific checkpoint protein could serve as a suitable therapeutic target for TNBC. Moreover, one of the processes in charge of controlling the expression of PD-L1 is the disappearance of PTEN. Consequently, agents that specifically target the PI3K pathway are expected to enhance antitumor immune responses, thereby potentially improving the efficacy of therapy for TNBC [70]. Immunotherapy in BC may face obstacles such as low immunogenicity, limited T-cell infiltration, and increased immunosuppression within the tumor microenvironment, which could hinder its effectiveness [71].

Figure 3: A schematic representation of the most recent breast cancer therapies. The sort and stage of malignancy have historically determined the type of therapy that is used. Combinations of several therapy modalities have also been employed more recently. Here is an outline of some of the most common therapy types and combination regimens.

Table 2: Current strategies of immunotherapy and cancer stem cell targeting in breast cancer.

Breast cancer and the function of cancer stem cells

The main reasons why cancer treatments fail include recurrence, heterogeneity, metastasis, resistance to radiation therapy and chemotherapy, and evasion of immune surveillance. All of these problems might be attributed to the characteristics of CSCs. CSCs can result in cancer recurrence, metastasis, resistance to multiple drugs, and radiation resistance due to their ability to stop at the G0 phase and produce new tumors. CSCs  may thus be among the most appealing targets for cancer treatment [80]. The first solid proof of CSC existence was presented by Bonnet and Dick in 1997. They demonstrated that only CD34+CD38− cells isolated from patients with acute myeloid leukemia (AML) might result in hematological malignancies in non-obese diabetic/severe combined immunodeficient mice [88]. According to the CSC theory, they are in charge of the development, growth, and metastasis of tumors as well as treatment resistance. By selectively destroying active tumor cells, traditional cancer treatments like chemotherapy and radiation therapy may shrink tumors. They overlook CSCs that are inactive, which leads to relapse and resistance, which may even make CSCs more resistant [81]. Recently, a number of innovative therapeutic approaches have been developed for eliminating CSCs and altering the environment (niches) in which these cells are supported. Subtle changes in surface markers and alterations in signaling cascades make them attractive therapeutic targets. Researchers have identified several potential therapeutic targets for these cells, including the ABC transporter superfamily, DNA repair and detoxification enzymes, anti-apoptotic proteins, and oncogenic signaling cascades such as Hedgehog, Notch, EGFR, and Wnt/β-catenin pathways. While certain treatment modalities are now successful in the removal of CSCs, others continue to be tested in clinical and preclinical environments [89] (Fig. 4). Some malignant cells are conceptually comparable to stem cells because they’re capable of self-renewal and produce sizable numbers of nontumor cells. To clarify the genesis of BC cells, two theories have been put forth: either individual mammary cells may be reprogrammed, or the regulation of progenitor cells or MaSCs can be altered. Both ideas are not restrictive of one another, and both entail the accumulation of distinct mutational occurrences. CSCs, which are found in BC, have been linked to the development, course, and relapse of BC. Recent developments in single-cell sequencing have made it simpler to decipher the clonal evolution inside each breast tumor. Nevertheless, there haven’t been many therapy studies conducted on this specific group of cancer cells [1]. The development of metastasis, drug resistance, and BC recurrence are all dependent on BC stem cells [86]. Recent studies have shown that CSCs are essential for targeting BC targets with treatment. With an emphasis on cancer immunotherapy, novel techniques for modifying CSCs as a targeted treatment for BC have been put forth [82]. There could be CSCs, a fraction of cells capable of self-renewal, division, and both the onset and progression of tumor development, according to recent data. Moreover, it is believed that CSCs are responsible for drug resistance, metastasis, and cancer recurrence. Therefore, medications that target breast CSCs could be more successful medicines for managing metastases and recurrences as well as treating BC. Natural compounds, particularly phytochemicals, have garnered a lot of interest lately because of their key signaling pathways, wide immunological profile, and capacity to target many kinds of malignancies [83]. CSCs are a tiny but significant fraction of unidentified tumor cells, making up about 0.1–1% of total tumor cells. Breast cancer stem cells (BCSCs) have been shown to exist and play a part in the development, spread, progression, resistance to therapy, and recurrence of tumors. Over the past ten years, significant strides have been achieved in the identification, description, and isolation of this particular kind of cell in the research of BCSCs. Growing knowledge of the molecular elements and signaling pathways that sustain BCSC and its stem-like characteristics has made it easier to create targeted therapeutics for BCSCs. Currently available therapies for BCSC subpopulations focus on chemicals that disrupt the resting state of BCSCs and make them chemoresistant and radiation-sensitive, and they also concentrate on BCSC signaling pathways and constituents that preserve BCSC characteristics [84]. When exposed to hypoxia, Aldh1a1 expression is associated with increased HIF-1α expression in BCSCs, but not HIF-2α [90]. Stem cells function in a variety of ways when used to treat malignancies. One of the processes that allows hematopoietic stem cells (HSCs) to rapidly migrate to designated stem cell niches in the bone marrow is homing. After this, the grafts undergo a homing phase that is essential before specialized blood cell formation. The manufacturing of the matrix-degrading enzyme MMP-2 and the engagement of the stem cell receptor CXCR4 with endothelial cells through VLA-4/5, LFA-1, and CD44 are necessary for this process. It additionally relies on these receptors being in active contact. The tumor-tropic effect, which occurs when mesenchymal stem cells (MSCs) move into the tumor microenvironment in response to the production of CXCL16, CCL-25, SDF-1, and IL-6 by tumor cells and then differentiate into tumor-associated types of cells, further promotes tumor-stroma development [91]. Only by effectively resolving the challenges associated with the accurate and effective targeting of BCSCs can BC be eradicated. The majority of BCSC markers are not appropriate for targeting since they are also expressed in normal stem cells, despite the fact that they are broadly accessible. Of these markers, CD44 is the most prevalent one for cancer stem cells and is crucial for preserving stemness. Anti-CD44 antibodies have been successful in encouraging the terminal differentiation of CSCs in spite of a number of issues related to post-translational modification and CD44 splicing. Consequently, there is a notable decrease in both the development of tumors and the occurrence of metastases [9295]. The strategy of using drugs that disrupt the self-renewal process of specific stem cells is a preferred approach, although it poses a significant obstacle due to the common pathways between normal and tumor stem cells. The introduction of the CSC hypothesis will revolutionize our understanding of breast carcinogenesis and provide a rationale for the classification, detection, and prevention of this disease. In the case of metastatic BC, the implications are likely to be even broader, as no definitive treatment is currently available for this advanced stage of the disease. Recent research studies have demonstrated functional links between metastasis, stem cells, and cancer cell survival [96]. According to the CSC model, tumors are composed of a cellular hierarchy in which only CSCs have the ability to proliferate indefinitely, giving rise to other tumor cells that lack this ability. An increasing corpus of data supports this notion by indicating that CSCs may be linked to a worse prognosis by causing recurrence, metastasis, and treatment failure [97]. Preclinical models should incorporate elements that target quiescent CSCs that persist locally or as micrometastases, even though surgery and supportive therapies are the first-line treatments for early-stage BC. This will help ensure the design of appropriate clinical trials [98]. Given the interconnectedness of the regulatory circuits controlling CSCs, eliminating this cell type may need a combination of targeted drugs [99]. Multidrug resistance, metastasis, radiation resistance, and recurrence of cancer are all triggered by CSCs since they have the ability to stop at G0 and produce new tumors. Therefore, one of the most promising therapies for treating cancer is the utilization of CSCs [80]. Because CSCs are extremely aggressive parts of the illness, TNBC represents one of the hardest cancers to treat. A new therapeutic approach might significantly improve TB treatment efficacy and survival rates while lowering the chance of a tumor recurrence [100].

Figure 4: Numerous therapies are aimed at eradicating cancer stem cells.

Probiotics: Definition and Special Role in Breast Cancer

The phrase “probiotics” has been around for a while, and Nobel laureate Eli Metchnikov recognized their immunomodulatory properties over a century ago. Probiotics are defined by the WHO and Food and Agriculture Organization (FAO) as living microorganisms (bacteria or yeasts) that improve the host’s health when administered in sufficient quantities (a dietary supplement in food)(101). Probiotics are a variety of microorganisms, particularly lactic acid bacteria (LABs). Most species of Lactobacillus, several species of Bifidobacteria, Enterococcus, and Streptococcus are considered to be lactic acid bacteria. Escherichia coli Nissle 1917, a non-laboratory probiotic, was put to use for the treatment of intestinal infectious diseases. These bacterial species are mostly found in the human stomach. Only Saccharomyces boulardii, a non-pathogenic probiotic yeast, is utilized. Probiotics, both laboratory and non-laboratory, are widely recognized as harmless organisms that can be utilized for veterinary or medical applications without risk. Probiotics’ quantitative cytotoxic action makes them suitable as natural cancer treatment agents. Additionally, probiotics generate certain growth inhibitors that only affect cancer cells.  A comprehensive overview of probiotics in breast BC therapy, organized by strain, biological activity, mechanisms, and evidence, is detailed in Table 3.  It has been demonstrated that Bifidobacterium animalis, Lactobacillus acidophilus, Lacticaseibacillus paracasei, and Bifidobacterium bifidum prevent cancer cells from proliferating in MCF7 cells. Lactobacillus helveticus R389 was implicated in enhancing immunological enhancement, cytokines (such as IL-10 and IL-4), and reducing tumor cell development upon subcutaneous injection of 4T1 mouse breast adenocarcinoma cells into the BALB/c mice model [102]. Probiotics seem to have potential uses in the prevention, treatment, and management of problems related to BC. Their metabolic properties, immunomodulatory effects, and ability to disrupt the estrogen enterohepatic cycle can be leveraged for preventive benefits [103]. Because probiotics like Lactobacillus have an impact on gut microbiota and systemic immunoregulatory function, they may offer protection against BC. Studies conducted in vitro have demonstrated that probiotic treatment causes cancer cells to undergo apoptosis and stops them from proliferating. Probiotic therapy has been demonstrated in animal models to prevent tumor growth, shrink tumors, and have other positive effects [104]. Numerous investigations have demonstrated that probiotics’ metabiotics have anti-inflammatory and antioxidant properties, which underpin their anticancer benefits. Han et al. investigated the anti-inflammatory characteristics of the Lactococcus lactis NK34 strain in RAW 264.7 cells. Both cell growth and the generation of proinflammatory cytokines and nitric oxide were significantly reduced. The bacteria Limosilactobacillus fermentum SNR1 and Limosilactobacillus mucosae AN1 significantly reduced paw edema and increased the production of the anti-inflammatory cytokine IL-10 in comparison to the proinflammatory cytokine IL-6. Certain strains of Lacticaseibacillus rhamnosus, Lacticaseibacillus paracasei, and Limosilactobacillus fermentum have significant antioxidant activity, according to Chooruk et al. In another investigation, Yang et al. discovered that probiotics downregulate the metabolic enzymes that generate reactive oxygen species (ROS), glutathione (GSH), and butyrate. Probiotics may produce folate and protein bacteriocin with a low molecular weight, which has anticancer, anti-inflammatory, and immunomodulatory properties, according to an earlier study [105]. Probiotics are being suggested as possible strategies for triggering apoptosis, causing cell-cycle arrest, and preventing BC cell growth [106].

The effects of probiotics on BC were investigated in a number of  in vivo  and in vitro  experiments. Moreno De Le Blanc et al. demonstrated that milk fermented by Lactobacillus helveticus R389 could control the immunological response in the mammary glands when localized BCs were present [162]. In mouse serum and mammary cells, rats fed and injected BC tumor cells fermented with milk Lactobacillus helveticus R389 show increased levels of IL-10 and lower levels of IL-6 cytokine, which inhibits breast tumor cells. In this regard, Yazdi and associates examined how oral treatment of Lactobacillus acidophilus affected the immune responses of BALB/c mice that received a BC cell transplant. The researchers suggest that a daily dosage of Lactobacillus acidophilus can increase the production of the immune-modulating cytokine IL-12 in splenocyte culture, even while the rate of tumor development in mice has reduced. Lacritz and associates showed that oral administration of the probiotic Limosilactobacillus reuteri prevented the early stages of BC in two mouse models: one with a genetic susceptibility to the disease, and the other fed a Western-style diet intended to cause mammary tumors. Additionally, Kassayová et al. observed that long-term treatment of Lactobacillus plantarum LS/07 efficiently combated BC through immunomodulation pathways [123]. In addition to medications, genes, and imaging agents that can identify tumors, recent research has demonstrated that NPs offer a number of benefits for tumor diagnosis and treatment [108]. In enough doses, probiotics—live, non-pathogenic microorganisms, have a significant positive impact on the health of the host. Whatever this definition, the reaction of dead probiotics and their metabolic secretions to various biological activities is equivalent to or greater than that of living probiotics. Acid resistance is one of probiotics’ primary qualities. Bile tolerance, mucous or epithelial cell adhesion resistance to antibiotics, the bile salt’s hydrolase potential for immunological stimulation, anti-carcinogenic and anti-disinfective properties against infections [109]. While the cytotoxic effects were less noticeable in normal HFF cells, the Bacillus coagulans supernatant

showed time- and concentration-dependent inhibitory effects on MCF 7 cells. The activation of apoptosis in malignancies was confirmed by accompanying flow cytometry data, downregulated expression of the anti-apoptotic BCL2 gene, and increased expression of the caspase-3 and caspase-9 genes [110]. It was discovered that Saccharomyces boulardii supernatant (SBS) had a stronger anticancer impact on MCF-7/mx cells than MCF-7. For the management of this kind of cancer in people, SBS is recommended as a possible anticancer medicine. It is highly advised that more research be done, particularly in vivo research, to discover more anticancer mechanisms of SBS against this malignancy. According to Zhang et al., Lacticaseibacillus casei and Lactobacillus acidophilus generate compounds that prevent MCF7 cell lines from proliferating [111]. In a metastatic mouse model of cancer, Levilactobacillus brevis (L. brevis) supplemented with selenium NPs decreased liver metastases and increased survival, according to Yazdi et al. In BALB/c mice with tumors, it also effectively stimulates immunological responses. Increased NK cell activity and greater quantities of T helper 1 cytokines, including IL-17 and IFN-γ, were among the immunological reactions [112].

Table 3: Probiotics in breast cancer prevention and therapy.