Systemic treatment in TNBC radical setting is recommended for tumors larger than 5 mm or node-positive disease. Preferred regimens involve anthracycline-based and taxane-based therapies, capecitabine and, in some cases, novel agents in the (neo)adjuvant setting (NCCN Clinical Practice Guidelines in Oncology, 2023; Triple-negative Breast Cancer, 2023).

In patients with metastatic endocrine-independent HER-2-negative BC with minor cancer-related symptoms and with a limited tumor burden, sequentially used single-agent schemes are preferred (Cardoso et al., 2020; NCCN Clinical Practice Guidelines in Oncology, 2023). The order of agents is not established and depends on numerous factors, for example, previous treatment received in adjuvant and metastatic setting, the risk of cross-resistance, Breast Cancer gene 1/2 (BRCA1/2) mutation status, or comorbidities. Typical options include (Cardoso et al., 2020; NCCN Clinical Practice Guidelines in Oncology, 2023; ER-positive HER2-negative, Breast Cancer, 2023; Triple-negative Breast Cancer, 2023):

• Taxanes: docetaxel, paclitaxel or nanoparticle albumin-bound (nab)paclitaxel.

Other agents used include:

• The Poly (ADP-ribose) polymerase (PARP) inhibitor olaparib in BRCA1/2 mutated patients.

For patients who require combination regimens due to rapidly progressing disease, symptoms or large tumor burden, there are numerous drug combinations available (Cardoso et al., 2020; NCCN Clinical Practice Guidelines in Oncology, 2023; ER-positive HER2-negative, Breast Cancer, 2023; Triple-negative Breast Cancer, 2023):

• Anthracycline-based schemes (with taxanes, cyclophosphamide and fluorouracil).

Numerous of the above-mentioned drugs have been derived from natural sources as secondary metabolites (Michalak and Püsküllüoğlu, 2022; Püsküllüoğlu and Michalak, 2022; Siddiqui et al., 2022). ADC such as T-DXd and SG, used in the discussed indication, can also be included in this group. The drugs conjugated with mAbs (trastuzumab directed against HER-2 for T-DXd and sacituzumab directed against Trophoblast cell-surface antigen 2 [TROP-2] for SG) are camptothecin derivatives (deruxtecan for T-DXd and SN-38 for SG) (Corti et al., 2021).

There is a long history of folk medicine with the usage of species being a source for anticancer naturally-derived agents. For example, the Himalayan yew (Taxus wallichiana Zucc., Taxaceae) bark and leaves are used in traditional medicine for steam baths (rheumatism), paste application (fractures, headaches), and medicinal hair oils. In Pakistan, stem decoction treats tuberculosis. Unani medicine uses them for Zarnab, addressing various conditions, while Ayurveda employs young shoots for a tincture treating headache, feeble pulse, giddiness, diarrhea, and severe biliousness (Juyal et al., 2014). Within the Ayurvedic medicinal system, various metabolites of periwinkle (Catharanthus roseus (L.) G. Don, Apocynaceae) are employed in traditional herbal medicine to address a range of health conditions, including diabetes, cancer, stomach disorders, liver, kidney and cardiovascular diseases (Kumar et al., 2022). The extract containing podophyllotoxin has been historically noted for its effectiveness as a laxative and as a treatment for diverse medical conditions, including tuberculosis, gonorrhea, menstrual disorders, dropsy, psoriasis, syphilis or venereal warts (Shah et al., 2021).

The main groups of secondary metabolites reported to have anticancer properties in endocrine-independent HER-2-negative breast cancer are phenolics, alkaloids, and terpenoids (Raman et al., 2018; Ding et al., 2020; Lee et al., 2020; Mazzei et al., 2020; Maungchanburi et al., 2021; Noh et al., 2021; Lin et al., 2022; Qi et al., 2022; Sancha et al., 2022; Youssef et al., 2022; Thilagavathi et al., 2023). Phenolics include simple phenols (phenolic acids and coumarins) and polyphenols (flavonoids and non-flavonoids such as tannins, lignans, and stilbenes). Examples of alkaloids, low-molecular-weight nitrogenous metabolites, are atropine, caffeine, capsaicin, cocaine, daturin, hiosciamin, lysergic acid, nicotine, strychnine, quinine (Alamgir, 2018; Thilagavathi et al., 2023). Terpenoids or terpenes, the major metabolites of essential oils, are classified as hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), sesterterpenes (C25), triterpenes (C30), and tetraterpenes/carotenoids. This group of secondary metabolites includes aromatic oils, carotenoids, resins, steroids, waxes, and others (Alamgir, 2018; Thilagavathi et al., 2023).

The key stage of research is the selection of biomass, which is a potential source of these anticancer metabolites. Subsequently, appropriate extraction techniques should be used to isolate metabolites of interest. In addition to simple extraction techniques, such as mixing/shaking, maceration or continuous extraction in the Soxhlet apparatus, novel extraction techniques, such as supercritical fluid extraction, sonication, ultrasound- and microwave-assisted extraction (Shin et al., 2017; Raman et al., 2018; Ding et al., 2020; Lee et al., 2020; Mazzei et al., 2020; Noh et al., 2021; Youssef et al., 2022) are in demand. The extraction process is usually carried out at room temperature in order to protect biologically active metabolites from degradation. Extracts derived from the biomass typically provide low yields of bioactive chemicals, which makes their isolation and purification time-consuming and expensive (Mazzei et al., 2020; Lin et al., 2022). Therefore, the selection of the appropriate extraction process, experimental conditions, and solvent seems to be of paramount importance. Finally, a detailed characterization of the obtained extract, which is a concentrate of biologically active metabolites, is required, or the isolation of metabolites from the extract showing desired anticancer properties specific to a given cancer type. The overriding goal is to search for extracts rich in bioactive substances that have an inhibitory effect on the proliferation and viability of cancer cells, while being comparatively safe for normal cells (Raman et al., 2018). Therefore, further fractionation of the extract is needed using the column chromatography separation. The purity of the active metabolites can be confirmed by a High-Performance Liquid Chromatography (HPLC). For the identification of different metabolites within an obtained fraction, gas chromatography coupled with mass analysis (GC-MS) can be applied. The functional groups in the isolated metabolites can be determined by Fourier Transform Infrared (FT-IR) Spectroscopy and the structure can be identified using Proton Nuclear Magnetic Resonance Spectroscopy (1H NMR) and other spectroscopic data (Maungchanburi et al., 2021; Heinrich et al., 2022; Qi et al., 2022; Sancha et al., 2022; Youssef et al., 2022).

The conducted research shows that not all extracts/extracted metabolites are cytotoxic to tested BC cell lines (Supplementary Table S1). For example, alliacane sesquiterpenes–purpuracolide B and purpuracolide C extracted from edible fungus Gomphus purpuraceus (Iwade) K. Yokoy. 1989 are inactive against MDA-MB-231 cell line (He et al., 2022). This shows how time-consuming and tedious it can be to work on the selection, extraction and testing of a metabolite that may have cytotoxic properties against cancer cells. Another challenge is the selectivity of the natural extracts/extracted metabolites produced in relation to the tested cancer cell lines. For example, Alsaraf et al. (2019) showed that the methanolic extract produced from plantain (Plantago lanceolata L., Plantaginaceae) leaves significantly inhibits the proliferation of TNBC CAL51 cells but demonstrate minor effect on the MDA-MB cells (Alsaraf et al., 2019). It also indicates that the heterogenicity of BC or even BC subtypes like TNBC cannot be neglected (Marra et al., 2020).

In the future, it is crucial to conduct comprehensive characterization of the obtained extracts to determine their chemical composition and to identify specific metabolites possessing anticancer properties. Some studies have already conducted fractionation of natural extracts and identified metabolites (e.g., pancratistatin (Youssef et al., 2022), lupeol (Sánchez-Valdeolívar et al., 2020), oleuropein aglycone (Zwartsen et al., 2019; Mazzei et al., 2020;), pyoluteorin (Ding et al., 2020), atranorin (Harikrishnan et al., 2021)) showing potential activity against endocrine-independent HER-2 negative hormone-pretreated BC.

Last years have yielded numerous publications regarding in vitro studies on natural metabolite derivatives in TNBC, but also a certain amount of research in HER-2 negative endocrine-pretreated BC. Currently, the majority of promising metabolites are derived from plants (e.g., phytochemicals), but fungi and algae/seaweeds are tempting sources as well (Supplementary Table S1).

An overview of numerous effects of natural metabolites in TNBC and HER-2 negative endocrine-resistant BC indication is presented in Figure 2. Most registered chemotherapeutic agents used in BC treatment act through inhibition of mitosis, lead to cell cycle arrest, influence deoxyribonucleic acid (DNA) replication processes, and thus induce apoptosis through well-known mechanisms (see Table 1).

The tumor microenvironment holds significant importance in pre-clinical research on naturally-derived agents. It consists of a complex interplay of cellular and non-cellular components surrounding the tumor, including immune cells, extracellular matrix, signaling molecules or blood vessels. These factors profoundly influence tumor growth, invasion, and how the tumor responds to various agents.

When exploring mechanisms related to tumor microenvironment role in endocrine-independent HER-2 negative breast cancer antiangiogenic activity (Hsu et al., 2020; Zunica et al., 2021), metabolism shift (Lin et al., 2019; Zhao et al., 2020; 2021; Deng et al., 2021; Chen et al., 2022; Fouzat et al., 2022; Lv et al., 2022) and immunomodulation (Guerra et al., 2021; Yang et al., 2021; Chen et al., 2022; Ke et al., 2022) (Figure 2) are presented.

Natural metabolites effects on cellular or molecular level in BC treatment are also explored in many other aspects than simply using them as potential drugs, e.g., for chemoprotection. One example is protection of hematopoiesis by extract derived from a perennial plant–Polygonatum sibiricum Redouté (Liliaceae) (Xie et al., 2021). Polysaccharides contained in this herb are claimed to inhibit activity of myeloid cells in immune tumor infiltrating cells and in spleen (in in vivo studies with mice with TNBC) at the same time protecting bone-marrow hematopoiesis (Xie et al., 2021). Other effects include: deactivating the stromal microenvironment by reducing tumor associated fibroblasts (Xu et al., 2020), reversing resistance to chemotherapy (Wang et al., 2020; Yi et al., 2021), and acting on cancer stem cells (Wang et al., 2020; Ke et al., 2022). Some natural metabolites do not act as medications by themselves, but increase sensitivity to currently available treatment options. In the case of hormone-pretreated tumors the efforts are directed at overcoming the endocrine resistance (e.g., Wang et al., 2021). Resveratrol overcomes endocrine resistance in tamoxifen-resistant MCF-7/TR cells by influencing the transforming growth factor-beta (TGF-β)/Smad signaling pathway (Shi et al., 2013). or TNBC tumors studies are aimed at chemotherapy resistance mechanisms (e.g., (Suarez-Arroyo and Martinez-Montemayor, 2018)). Natural raw materials are also known to serve as carriers of many secondary metabolites that have been reported to have anticancer properties in both in vitro and in vivo experiments, especially metabolites belonging to phenolics, alkaloids, and terpenoids (Püsküllüoğlu and Michalak, 2022).

With all the enthusiasm accompanying these metabolites there is always a risk of increased toxicity or drugs interaction, e.g., in research by Zunica et al. (2021) moringa seed extract (Moringa oleifera Lam., Moringaceae) used in in vivo experiment on mice with TNBC and obesity has shown a negative interaction when used as a concurrent systemic treatment (Zunica et al., 2021).

Nanocarriers offer enhanced drug activity and utility through controlled release, extended circulation, and targeted delivery to cancer cells. Encapsulating drugs in nanoparticles can enable alternative administration routes. These nanocarriers utilize passive and active targeting mechanisms. Among others, nanoforms of taxanes, anthracyclines, camptothecins or eribulin mesylate have recently been tested (Yap et al., 2021; Miguel et al., 2022; Püsküllüoğlu and Michalak, 2022). Nanoparticles can be obtained from natural sources by themselves acting as immunomodulators, drugs, or in cancer prevention or cytoprotection (Püsküllüoğlu and Michalak, 2022). Next to clinically applied nanoforms of drugs new options are tested in pre-clinical and clinical setting (see below). Example of recently published pre-clinical study involves doxorubicin-loaded magnetite nanoparticles in TNBC in vitro cell line model (Markhulia et al., 2023).

Currently, the greatest group of targeting agents seems to be ADCs with SG and T-DXd being already approved in this indication (Cardoso et al., 2020; Püsküllüoğlu et al., 2023). ADC selectively recognizes and binds to specific cancer cells. The linker acts as a bridge, connecting the antibody to the cytotoxic payload, which exerts its effect by inducing cell death in the targeted cancer cells. The phenomenon where neighbouring cancer cells, not directly targeted by the drug, are still affected by the cytotoxic payload is called bystander effect. Ideal ADC payloads should have high cytotoxicity, low immunogenicity, and high stability. They should also possess modifiable functional groups for conjugation, promote bystander killing effects, have proper water solubility, and target intracellular sites for effective tumor cell penetration (Püsküllüoğlu et al., 2023; Wang et al., 2023). ADCs including naturally-derived agents that are tested in clinical trials in endocrine-independent HER-2-negative BC are presented in the next chapter. For pre-clinical setting a good example is ADC consisting of humanized MUC1 antibody linked to monomethyl auristatin (MMAE) and showing activity in cell line model (Li et al., 2023). MMAE is an antimitotic agent derived from sea hare (Dolabella auricularia [Lightfoot], 1786) from Aplysiidae that is too toxic to be used on its own, but linking to mAb allows toxicity to be limited (Püsküllüoğlu and Michalak, 2022).

It is expected that both: new receptors and new natural metabolites linked to targeting part will be further explored in the nearest future. The emphasis will be placed on the exploration of more specific targets for these BC populations. Exosome-based carriers are another alternative for modern drug delivery system (Song et al., 2021). However, the implementation of exosomes in BC clinical settings has not yet been realized, resulting in an uncertain assessment of their usefulness. Examples of other options used to target cytotoxic agents include targeted small molecules (Sun et al., 2021).

Figure 3 presents an overview of already applied and potential delivery strategies for natural metabolites in endocrine-independent HER-2 negative BC.

Undoubtedly, the highest expectations are placed upon this particular group of studies. The pursuit of novel pharmaceutical agents exhibiting no cross-resistance with previously employed treatments and enhancing overall survival stands as the paramount hope in the realm of clinical research. Challenges may include lack of production standardization, limited access to natural sources, and thus, cost of the treatment. Some naturally-derived medications even when tested in clinical trials have no clear definition of their components or the solvent. Efforts to better control this issue are imperative, as it can lead to challenges in reproducing clinical data, as well as pose a significant risk of drug interactions. NCT04403529is the phase 3 randomized, double-blind trial checking the efficacy of Traditional Chinese Medicine in TNBC adjuvant setting. Primary endpoints are disease free survival (DFS) and quality of life. Unfortunately, clinicaltrial.gov web page devoted to that study does not explain which twelve herbal metabolites are incorporated into that medicine. That issue is a certain limitation in few ongoing clinical trials. In the phase 4 NCT02615457 trial, Huaier granules are being evaluated for their efficacy in radically treated TNBC. Huaier granules are a traditional Chinese medication extracted from a mushroom Trametes robiniophila Murrill 1907 (Polyporaceae) applied in colitis, nephrosis, tuberous sclerosis, and various cancers (Narayanan et al., 2023). Following international guidelines all tumors greater than 5 mm should receive preoperative systemic treatment (NCCN Clinical Practice Guidelines in Oncology, 2023). As per protocol patients who completed neoadjuvant treatment are excluded. As a result, a very low recruitment rate or comparison to control arm receiving the treatment inferior to current standard of care can be expected. This is another limitation of these clinical trials. The phase 1/2 NCT03387085 trial in metastatic TNBC setting tests metronomic chemotherapy of numerous naturally derived agents in terms of safety and ORR. Drugs such as: albumin-binding prodrug of doxorubicin; a set of vaccines derived from recombinant Saccharomyces cerevisiae yeast; nab-paclitaxel and other chemotherapeutics and biological agents are included in that trial. The phase 2 NCT05007444 study with P2Et (a standardized extract of tara (Caesalpinia spinosa (Molina) Kuntze, Fabaceae) in addition to standard neoadjuvant therapy is being performed on BC patients (not eligible for anti-HER-2 treatment) in order to assess optimal biological dose of P2Et based on its toxicological profile and other parameters as secondary endpoints. The phase 2 NCT05403333 trial is examining the efficacy (PFS) of weekly utidelone in HER-2 negative inoperable or metastatic BC (both TNBC and hormone-pretreated). This metabolite, a modified epothilone (see Table 1 for ixabepilone source) analog has already been tested in BC setting and although positive, the previous study did not result in drug approval (Xu et al., 2021a). An intriguing study, yet one that raises many unanswered questions and concerns (e.g., about the broad study population, which includes DCIS, and dosage, staging of the disease), is the phase 1 trial (NCT05680662) examining herbal titled “The Study of Quadruple Therapy Quercetin, Zinc, Metformin, and EGCG as Adjuvant Therapy for Early, Metastatic Breast Cancer, and Triple-negative Breast Cancer”. Quercetin, present in foods like kale, berries, onions, cherries, red grapes, broccoli, tea, and red wine, is traditionally used to prevent diseases such as osteoporosis, lung and cardiovascular diseases, and cancer. Epigallocatechin gallate (EGCG) is a polyphenol present in green tea has been tested for its chemopreventive activity (Du et al., 2012; Xu et al., 2019). Natural metabolites have been also tested in other contexts such as primary or secondary BC prevention and management of side effects. Epidiferphane, a claimed neuroregenerative dietary supplement (Steindler and Reynolds, 2017), is being evaluated alongside taxanes in BC to prevent peripheral neuropathy in the phase 1/2 NCT05074290 trial. Pentoxifylline, a synthetic xanthine derivative originally derived from coffee beans and tea leaves (Dastgheib et al., 2022). Unfortunately, most studies in this context are expected to yield negative results, as seen with soy protein for BC prevention in the NCT00204477 trial or PSC 833 (a cyclosporine derivative from the fungus Tolypocladium inflatum W. Gams 1971, Ophiocordycipitaceae) for multidrug-resistance inhibition in BC patients receiving taxanes (NCT00002826).

Studies regarding nanoforms of existing medications and drugs that are active, but too toxic to be used on their own are costly and more likely to fail than traditional ones. What is more, the benefits of introducing drug nanoform are difficult to prove in pre-clinical studies. Nonetheless, that direction is constantly proving to be effective in terms of obtaining better treatment outcomes, diminishing toxicity, offers better solubility or a new route for administration (Püsküllüoğlu and Michalak, 2022). In a single arm phase 2 the PHENOMENAL study (NCT03328884) nanoliposomal irinotecan is being tested for central nervous system (CNS) ORR in HER-2 negative BC patients with brain metastases. This drug is also being evaluated in combination with pembrolizumab in TNBC brain metastatic population in phase 2 NCT05255666 trial with CNS Disease Control Rate (DCR) as the primary endpoint. While nanoforms of irinotecan are already approved for use in other indications such as pancreatic cancer (Wang-Gillam et al., 2019) docetaxel’s nanoforms have no registration as of now. In phase 3 NCT03671044 study, nanosomal docetaxel lipid suspension and a standard docetaxel are compared for ORRs in later lines of palliative TNBC treatment. Paclitaxel’s nanoforms have been approved for use in few indications including metastatic BC or pancreatic cancer (Samaan et al., 2019). In phase 3 NCT04137653 study, nab-paclitaxel is being tested in radical treatment of TNBC.

Among potential benefits of ADC: selective toxicity, increased potency, reduced drug resistance can be named. The limitations in these trials are an industry-sponsored bias and a risk of setting endpoints that is not optimal, but easier to achieve. Additionally, endocrine-independent HER-2-negative breast cancer lacks specific therapeutic targets tailored to this subtype, potentially limiting the efficacy of tested ADCs. Patritumab Deruxtecan is a camptothecin derivative linked to anti-HER-3 mAb. In the phase 2 NCT04699630 trial, ORR and 6-month PFS are being assessed in metastatic BC including two arms for HER-2 negative endocrine-resistant patients. In another phase 2 NCT04965766 trial, the same drug is being tested for ORR in the metastatic HER-2 negative endocrine-resistant BC population. The phase 3 ANGLeD trial (NCT03613181) covers HER-2 negative metastatic BC patients with leptomeningeal disease and extremely unfavorable prognosis. Paclitaxel trevatide (paclitaxel-Angiopep-2 conjugate) is compared to other agents (including eribulin mesylate) to check for OS. In the phase 2 NCT04742153 trial MR002, an anti-HER2 mAb conjugated MMAE is being assessed for ORR in HER-2 low metastatic BC. The phase 1/2 NCT04441099 trial assesses the dose and anti-tumor activity of anthracycline-based ADC targeting Receptor tyrosine kinase-like orphan receptor 1 in few indications including metastatic TNBC (for the phase 2 part). The TROPION-PanTumor02 phase 1/2 (NCT05460273) trial explores the anti-TROP2 mAb linked to deruxtecan ADC (Datopotamab- DXd) in terms of ORR in different BC cohorts.

In number of trials, currently approved drugs are being tested to search for better administration scheme or set standards in their combination with other medicines or determine optimal therapy sequences. The main limitation is the retrospective observational design of these studies, driven by challenges in organizing randomized controlled trials (RCTs) for non-patented drugs, primarily due to financial constraints. Metronomic chemotherapy uses low doses of drugs at regular short intervals, with antiangiogenic and antineoplastic effects (Cazzaniga et al., 2021). For example, the phase 2 NCT03071926 trial explores the outcomes of metronomic pegylated doxorubicin in endocrine-resistant advanced BC, while phase 2 NCT05747326 study checks 1-year performance free survival (PFS) of capecitabine and vinorelbine in HER-2 negative BC. An example of study that tries to set the place for commonly used drugs in TNBC radical treatment setting is phase 4 NCT04136782 trial with carboplatin, nab-paclitaxel, epirubicin and docetaxel.

Among the studies exploring new combinations of known drugs, there are numerous examples: the KEYDOX phase 1/2 trial (NCT03591276) studies the efficacy–objective response rate (ORR) and safety of pembrolizumab (an anti-PD-1 mAb) with pegylated liposomal doxorubicin in endocrine-resistant BC. In NCT04039230 phase 1/2 study SG is tested in combination with talazoparib (a PARP inhibitor) in metastatic TNBC. The same ADC is tested together with PI3K inhibitor alpelisib in metastatic, pretreated HER-2 negative BC in the phase 1 ASSET trial (NCT05143229), and other ADC T-DXd is checked for its safety when administered with nivolumab (an anti-PD1 mAb) in phase 1 NCT03523572 study including patients with BC.

Improving clinical trials involving natural-source medications is crucial due to challenges like lack of standardization and limited access to natural sources. Ambiguity in drug components and solvents can hinder data reproducibility and pose drug interaction risks. Addressing these issues through rigorous quality control, standardized protocols, and drug characterization is imperative for more reliable and effective clinical trial outcomes. Improving clinical trials for nanoforms of existing medications and toxic drugs requires addressing cost challenges and optimizing pre-clinical data to enhance the chances of developing safe and effective nanoform treatments. Enhancing clinical studies for drugs composed of natural-source-derived metabolites attached to targeting part requires addressing industry-sponsored bias and carefully selecting clinically relevant endpoints. Transparent trial design, independent oversight, and adaptive approaches can enhance scientific validity and yield more reliable results. Collaborative funding, innovative trial designs, and real-world data utilization are potential approaches to address limitations in clinical trials for currently approved drugs when searching for better therapy sequence, administration scheme or drugs’ combinations.