Antioxidant Enzymes in Cancer Cells: Their Role in Photodynamic Therapy Resistance and Potential as Targets for Improved Treatment Outcomes.

Photodynamic therapy (PDT) is a selective tumor treatment that consists of a photosensitive compound-a photosensitizer (PS), oxygen, and visible light. Although each component has no cytotoxic properties, their simultaneous use initiates photodynamic reactions (PDRs) and sequentially generates reactive oxygen species (ROS) and/or free radicals as cytotoxic mediators, leading to PDT-induced cell death. Nevertheless, tumor cells develop various cytoprotective mechanisms against PDT, particularly the adaptive mechanism of antioxidant status. This review integrates an in-depth analysis of the cytoprotective mechanism of detoxifying ROS enzymes that interfere with PDT-induced cell death, including superoxide dismutase (SOD), catalase, glutathione redox cycle, and heme oxygenase-1 (HO-1). Furthermore, this review includes the use of antioxidant enzymes inhibitors as a strategy in order to diminish the antioxidant activities of tumor cells and to improve the effectiveness of PDT. Conclusively, PDT is an effective tumor treatment of which its effectiveness can be improved when combined with a specific antioxidant inhibitor.

Unmasking the Deceptive Nature of Cancer Stem Cells: The Role of CD133 in Revealing Their Secrets.

Cancer remains a leading cause of death globally, and its complexity poses a significant challenge to effective treatment. Cancer stem cells and their markers have become key players in tumor growth and progression. CD133, a marker in various cancer types, is an active research area as a potential therapeutic target. This article explores the role of CD133 in cancer treatment, beginning with an overview of cancer statistics and an explanation of cancer stem cells and their markers. The rise of CD133 is discussed, including its structure, functions, and occurrence in different cancer types. Furthermore, the article covers CD133 as a therapeutic target, focusing on gene therapy, immunotherapy, and approaches to affect CD133 expression. Nanoparticles such as gold nanoparticles and nanoliposomes are also discussed in the context of CD133-targeted therapy. In conclusion, CD133 is a promising therapeutic target for cancer treatment. As research in this area progresses, it is hoped that CD133-targeted therapies will offer new and effective treatment options for cancer patients in the future.

Drug affinity and targeted delivery: double functionalization of silk spheres for controlled doxorubicin delivery into Her2-positive cancer cells.

BACKGROUND: The optimal drug delivery system should be biocompatible, biodegradable, and allow the sustained release of the drug only after it reaches the target cells. Silk, as a natural polymer, is a great candidate for building drug carriers. Genetically engineered silks offer the possibility of functionalization. Previously, we characterized bioengineered silk spheres that were functionalized with H2.1 peptide that selectively delivered a drug to Her2-positive cancer cells. However, drug leakage from the silk spheres showed the need for improved control. RESULTS: To control the drug loading and release, we designed and produced functional silk (DOXMS2) that contains a DOX peptide with an affinity for doxorubicin. The DOXMS2 spheres showed the decreased release of doxorubicin compared with MS2 particles. Next, the DOXMS2 silk was blended with the H2.1MS1 polymer to improve the control of doxorubicin binding and release into Her2-positive cancer cells. The H2.1MS1:DOXMS2 particles showed the highest doxorubicin-loading capacity and binding per cell, which resulted in the highest cytotoxic effect compared with that of other sphere variants. Since drug release at a pH of 7.4 from the blended H2.1MS1:DOXMS2 particles was significantly lower than from blended spheres without DOXMS2 silk, this indicated that such particles could control the release of the drug into the circulatory system before the carrier reached the tumor site. CONCLUSIONS: This strategy, which is based on the blending of silks, allows for the generation of particles that deliver drugs in a controlled manner.

Functionalized silk spheres selectively and effectively deliver a cytotoxic drug to targeted cancer cells in vivo.

BACKGROUND: Chemotherapy is often a first-line therapeutic approach for the treatment of a wide variety of cancers. Targeted drug delivery systems (DDSs) can potentially resolve the problem of chemotherapeutic drug off-targeting effects. Herein, we examined in vivo models to determine the efficacy of Her2-targeting silk spheres (H2.1MS1) as DDSs for delivering doxorubicin (Dox) to Her2-positive and Her2-negative primary and metastatic mouse breast cancers. RESULTS: The specific accumulation of H2.1MS1 spheres was demonstrated at the site of Her2-positive cancer. Dox delivered only by functionalized H2.1MS1 particles selectively inhibited Her2-positive cancer growth in primary and metastatic models. Moreover, the significant effect of the Dox dose and the frequency of treatment administration on the therapeutic efficacy was indicated. Although the control MS1 spheres accumulated in the lungs in Her2-positive metastatic breast cancer, the Dox-loaded MS1 particles did not treat cancer. Histopathological examination revealed no systemic toxicity after multiple administrations and at increased doses of Dox-loaded silk spheres. Although the studies were performed in immunocompetent mice, the H2.1MS1 silk spheres efficiently delivered the drug, which exerted a therapeutic effect. CONCLUSION: Our results indicated that functionalized silk spheres that enable cell-specific recognition, cellular internalization, and drug release represent an efficient strategy for cancer treatment in vivo.

Silk as an innovative biomaterial for cancer therapy.

Silk has been used for centuries in the textile industry and as surgical sutures. In addition to its unique mechanical properties, silk possesses other properties, such as biocompatibility, biodegradability and ability to self-assemble, which make it an interesting material for biomedical applications. Although silk forms only fibers in nature, synthetic techniques can be used to control the processing of silk into different morphologies, such as scaffolds, films, hydrogels, microcapsules, and micro- and nanospheres. Moreover, the biotechnological production of silk proteins broadens the potential applications of silk. Synthetic silk genes have been designed. Genetic engineering enables modification of silk properties or the construction of a hybrid silk. Bioengineered hybrid silks consist of a silk sequence that self-assembles into the desired morphological structure and the sequence of a polypeptide that confers a function to the silk biomaterial. The functional domains can comprise binding sites for receptors, enzymes, drugs, metals or sugars, among others. Here, we review the current status of potential applications of silk biomaterials in the field of oncology with a focus on the generation of implantable, injectable and targeted drug delivery systems and the three-dimensional cancer models based on silk scaffolds for cancer research. However, the systems described could be applied in many biomedical fields.

Functionalized spider silk spheres as drug carriers for targeted cancer therapy.

Bioengineered spider silk is a biomaterial that combines the properties of self-assembly, biocompatibility and biodegradability with reasonable accessibility and a simple purification procedure. Moreover, genetic engineering enables the functionalization of silk by adding the peptide coding sequences of the desired attribute. Hybrids composed of Her2 binding peptides (H2.1 or H2.2) and bioengineered silk MS1 (based on the MaSp1 sequence from N. clavipes) were designed. Bioengineered silks were expressed in a bacterial system and purified using a tag-free thermal method. The hybrid silks with N-terminal functionalization were bound more efficiently to cells that were overexpressing Her2 than those with the C-terminal fusion. Moreover, the functionalization did not impede the self-assembly property of bioengineered silk, enabling the processing of silk proteins into spheres. The binding domains were exposed on the surface of the spheres, because the functionalized particles specifically bound and internalized into Her2-overexpressing cells. The binding of the functionalized spheres to Her2-positive cells was significantly higher compared with the control sphere and Her2-negative cell binding. Silk spheres were loaded with doxorubicin and showed pH-dependent drug release. The silk spheres were not cytotoxic, unless they were loaded with the drug doxorubicin. This study indicates the ability of drug-loaded functionalized spider silk spheres to serve as a carrier for targeted cancer therapy.

Endotoxin reduction from biotech silk material inhibits the production of anti-silk antibodies in mice.

Eliminating endotoxins is a common problem in the development of biotechnologically produced pharmaceuticals or biomaterials. Residual endotoxins in the final sample may hamper the properties of the product or induce severe adverse effects. Developing an effective downstream purification protocol that ensures a lack of minimal endotoxin content in the final product can be a challenging task. In our previous studies, we developed nanospheres produced from bioengineered silks. Despite their good overall biocompatibility, in vivo characterization of spheres showed mild activation of the immune system (mainly in terms of anti-silk antibody production). Herein, we examined, if the endotoxins delivered with the silk spheres might have contributed to activating the adaptive immune response. We investigated various commercially available methods for endotoxin removal that can be applied as an extra step in downstream endotoxin removal from MS1-type silk proteins. We selected a method that allowed for a 10-fold reduction of endotoxin content in soluble silk and 2-fold in the final product (silk spheres). The reduced level of endotoxins improved the biocompatibility of the silk spheres as these particles induced negligible titers of anti-silk antibodies in an in vivo immune study. Since endotoxins can enhance life-threatening immune responses, it is crucial to optimize the method of their removal before clinical use not only of silk-based products but also of other biomolecules produced biotechnologically.

MMPs-responsive silk spheres for controlled drug release within tumor microenvironment.

Silk is a biocompatible and biodegradable material that enables the formation of various morphological forms, including nanospheres. The functionalization of bioengineered silk makes it possible to produce particles with specific properties. In addition to tumor cells, the tumor microenvironment (TME) includes stromal, immune, endothelial cells, signaling molecules, and the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) are overexpressed in TME. We investigated bioengineered spider silks functionalized with MMP-responsive peptides to obtain targeted drug release from spheres within TME. Soluble silks MS12.2MS1, MS12.9MS1, and MS22.9MS2 and the corresponding silk spheres carrying MMP-2 or MMP-2/9 responsive peptides were produced, loaded with doxorubicin (Dox), and analyzed for their susceptibility to MMP-2/9 digestion. Although all variants of functionalized silks and spheres were specifically degraded by MMP-2/9, the MS22.9MS2 nanospheres showed the highest levels of degradation and release of Dox after enzyme treatment. Moreover, functionalized spheres were degraded in the presence of cancer cells releasing MMP-2/9. In the 2D and 3D spheroid cancer models, the MMP-2/9-responsive substrate was degraded and released from spheres when loaded into MS22.9MS2 particles but not into the control MS2 spheres. The present study demonstrated that a silk-based MMP-responsive delivery system could be used for controlled drug release within the tumor microenvironment.

Designer cytokine hyper interleukin 11 (H11) is a megakaryopoietic factor.

Interleukin-11 (IL-11) displays megakaryopoietic activity. We constructed super-cytokine Hyper- IL11 (H11) by linking soluble IL-11 receptor α (sIL-11Rα) with IL-11, which directly targets ß-receptor (gp130) signal transducing subunit. The effects of H11 on hematopoiesis with a focus on megakaryopoiesis were studied. The expansion, differentiation and type of colony formation of cord blood progenitor Lin-CD34+ cells were analyzed. H11 was more effective than recombinant human IL-11 (rhIL-11) in enhancement of the Lin-CD34+ cells expansion and differentiation into megakaryocytes (Mk). It induced higher expression of CD41a and CD61 antigens, resulting in a substantially larger population of CD34-CD41a(high)CD61(high) cells. H11 treatment led to increased number of small and mainly medium megakaryocyte colony formation (Mk-CFU). Moreover, it induced the formation of a small number of large colonies, which were not observed following rhIL-11 treatment. Significantly higher number of H11 derived Mk colonies released platelets-like particles (PLP). Furthermore, H11 was considerably more potent than rhIL-11 in promoting differentiation of Lin-CD43+ cells toward erythrocytes. Our results indicate that H11 is more effective than rhIL-11 in enhancing expansion of early progenitors and directing them to megakaryocyte and erythroid cells and in inducing maturation of Mk. Thus, H11 may prove beneficial for thrombocytopenia treatment and/or an ex vivo expansion of megakaryocytes.

Oligonucleotide-Based Therapeutics for STAT3 Targeting in Cancer-Drug Carriers Matter.

High expression and phosphorylation of signal transducer and transcription activator 3 (STAT3) are correlated with progression and poor prognosis in various types of cancer. The constitutive activation of STAT3 in cancer affects processes such as cell proliferation, apoptosis, metastasis, angiogenesis, and drug resistance. The importance of STAT3 in cancer makes it a potential therapeutic target. Various methods of directly and indirectly blocking STAT3 activity at different steps of the STAT3 pathway have been investigated. However, the outcome has been limited, mainly by the number of upstream proteins that can reactivate STAT3 or the relatively low specificity of the inhibitors. A new branch of molecules with significant therapeutic potential has emerged thanks to recent developments in the regulatory function of non-coding nucleic acids. Oligonucleotide-based therapeutics can silence target transcripts or edit genes, leading to the modification of gene expression profiles, causing cell death or restoring cell function. Moreover, they can reach untreatable targets, such as transcription factors. This review briefly describes oligonucleotide-based therapeutics that found application to target STAT3 activity in cancer. Additionally, this review comprehensively summarizes how the inhibition of STAT3 activity by nucleic acid-based therapeutics such as siRNA, shRNA, ASO, and ODN-decoy affected the therapy of different types of cancer in preclinical and clinical studies. Moreover, due to some limitations of oligonucleotide-based therapeutics, the importance of carriers that can deliver nucleic acid molecules to affect the STAT3 in cancer cells and cells of the tumor microenvironment (TME) was pointed out. Combining a high specificity of oligonucleotide-based therapeutics toward their targets and functionalized nanoparticles toward cell type can generate very efficient formulations.

A designer hyper interleukin 11 (H11) is a biologically active cytokine.

BACKGROUND: Interleukin 11 (IL-11) is a pleiotropic cytokine with anti-apoptotic, anti-inflammatory and hematopoietic potential. The IL-11 activity is determined by the expression of the IL-11R receptor alpha (IL-11Rα) and the signal transducing subunit ß (gp130) on the cell membrane. A recombinant soluble form of the IL-11Rα (sIL-11Rα) in combination with IL-11 acts as an agonist on cells expressing the gp130 molecule. We constructed a designer cytokine Hyper IL-11 (H11), which is exclusively composed of naturally existing components. It contains the full length sIL-11Rα connected with the mature IL-11 protein using their natural sequences only. Such a construct has two major advantages: (i) its components are as close as possible to the natural forms of both proteins and (ii) it lacks an artificial linker what should avoid induction of antibody production. RESULTS: The H11 construct was generated, the protein was produced in a baculovirus expression system and was then purified by using ion exchange chromatography. The H11 protein displayed activity in three independent bioassays, (i) it induced acute phase proteins production in HepG2 cells expressing IL-11, IL-11Rα and gp130, (ii) it stimulated the proliferation of B9 cells (cells expressing IL-11Rα and gp130) and (iii) proliferation of Baf/3-gp130 cells (cells not expressing IL-11 and IL-11Rα but gp130). Moreover, the preliminary data indicated that H11 was functionally distinct from Hyper-IL-6, a molecule which utilizes the same homodimer of signal transducing receptor (gp130). CONCLUSIONS: The biologically active H11 may be potentially useful for treatment of thrombocytopenia, infertility, multiple sclerosis, cardiovascular diseases or inflammatory disorders.

In vivo study of the immune response to bioengineered spider silk spheres.

Bioengineered MS1 silk is derived from major ampullate spidroin 1 (MaSp1) from the spider Nephila clavipes. The MS1 silk was functionalized with the H2.1 peptide to target Her2-overexpressing cancer cells. The immunogenic potential of drug carriers made from MS1-type silks was investigated. The silk spheres were administered to healthy mice, and then (i) the phenotypes of the immune cells that infiltrated the Matrigel plugs containing spheres (implanted subcutaneously), (ii) the presence of silk-specific antibodies (after two intravenous injections of the spheres), (iii) the splenocyte phenotypes and their activity after restimulation ex vivo in terms of proliferation and cytokine secretion (after single intravenous injection of the spheres) were analyzed. Although the immunogenicity of MS1 particles was minor, the H2.1MS1 spheres attracted higher levels of B lymphocytes, induced a higher anti-silk antibody titer, and, after ex vivo restimulation, caused the activation of splenocytes to proliferate and express more IFN-γ and IL-10 compared with the PBS and MS1 groups. Although the H2.1MS1 spheres triggered a certain degree of an immunological response, multiple injections (up to six times) neither hampered the carrier-dependent specific drug delivery nor induced toxicity, as previously indicated in a mouse breast cancer model. Both findings indicate that a drug delivery system based on MS1-type silk has great potential for the treatment of cancer and other conditions.

[Engineered spider silk: the intelligent biomaterial of the future. Part I]. / Inzynierowany jedwab pajeczy: inteligentny biomaterial przyszlosci. Czesc I.

The unique properties of spider silk such as strength, extensibility, toughness, biocompatibility and biodegradability are the reasons for the recent development in silk biomaterial technology. For a long time scientific progress was impeded by limited access to spider silk. However, the development of the molecular biology strategy was a breaking point in synthetic spider silk protein design. The sequences of engineered spider silk are based on the consensus motives of the corresponding natural equivalents. Moreover, the engineered silk proteins may be modified in order to gain a new function. The strategy of the hybrid proteins constructed on the DNA level combines the sequence of engineered silk, which is responsible for the biomaterial structure, with the sequence of polypeptide which allows functionalization of the silk biomaterial. The functional domains may comprise receptor binding sites, enzymes, metal or sugar binding sites and others. Currently, advanced research is being conducted, which on the one hand focuses on establishing the particular silk structure and understanding the process of silk thread formation in nature. On the other hand, there are attempts to improve methods of engineered spider silk protein production. Due to acquired knowledge and recent progress in synthetic protein technology, the engineered silk will turn into intelligent biomaterial of the future, while its industrial production scale will trigger a biotechnological revolution.

[Engineered spider silk: the intelligent biomaterial of the future. Part II]. / Inzynierowany jedwab pajeczy: inteligentny biomaterial przyszlosci. Czesc II.

The development and progress in engineered spider silk manufacturing has enabled its practical application. Recombinant spider silk can assemble in several morphological forms such as films, hydrogels, fibers, scaffolds, microcapsules, and micro- and nanospheres. The in vitro fiber formation takes place by mimicking the natural spinning process in the spider spinning gland: in the presence of phosphate ions and dragging forces. Films are obtained by evaporation of solvent from the silk solution, while the result of evaporation of the solvent in the presence of porogens is a silk scaffold. Hydrogels are formed by spontaneous polymerization of silk particles in solutions at low pH. The silk film assembled at the interface of two immiscible phases forms microcapsules. The smallest of the described forms--silk spheres--are obtained by salting out the silk protein solution after addition of the phosphate ions. Common properties of the silk biomaterials are biocompatibility and biodegradability, which make them suitable for a number of applications in medicine and pharmacy. Moreover, the strategy of hybrid proteins which provides the desired function to biomaterial will further expand their potential use.

Systemic and Local Silk-Based Drug Delivery Systems for Cancer Therapy.

For years, surgery, radiotherapy, and chemotherapy have been the gold standards to treat cancer, although continuing research has sought a more effective approach. While advances can be seen in the development of anticancer drugs, the tools that can improve their delivery remain a challenge. As anticancer drugs can affect the entire body, the control of their distribution is desirable to prevent systemic toxicity. The application of a suitable drug delivery platform may resolve this problem. Among other materials, silks offer many advantageous properties, including biodegradability, biocompatibility, and the possibility of obtaining a variety of morphological structures. These characteristics allow the exploration of silk for biomedical applications and as a platform for drug delivery. We have reviewed silk structures that can be used for local and systemic drug delivery for use in cancer therapy. After a short description of the most studied silks, we discuss the advantages of using silk for drug delivery. The tables summarize the descriptions of silk structures for the local and systemic transport of anticancer drugs. The most popular techniques for silk particle preparation are presented. Further prospects for using silk as a drug carrier are considered. The application of various silk biomaterials can improve cancer treatment by the controllable delivery of chemotherapeutics, immunotherapeutics, photosensitizers, hormones, nucleotherapeutics, targeted therapeutics (e.g., kinase inhibitors), and inorganic nanoparticles, among others.

Hyperthermia treatment of cancer cells by the application of targeted silk/iron oxide composite spheres.

Magnetic iron oxide nanoparticles (IONPs) are one of the most extensively studied materials for theranostic applications. IONPs can be used for magnetic resonance imaging (MRI), delivery of therapeutics, and hyperthermia treatment. Silk is a biocompatible material and can be used for biomedical applications. Previously, we produced spheres made of H2.1MS1 bioengineered silk that specifically carried a drug to the Her2-overexpressing cancer cells. To confer biocompatibility and targeting properties to IONPs, we blended these particles with bioengineered spider silks. Three bioengineered silks (MS1Fe1, MS1Fe2, and MS1Fe1Fe2) functionalized with the adhesion peptides F1 and F2, were constructed and investigated to form the composite spheres with IONPs carrying a positive or negative charge. Due to its highest IONP content, MS1Fe1 silk was used to produce spheres from the H2.1MS1:MS1Fe silk blend to obtain a carrier with cell-targeting properties. Composite H2.1MS1:MS1Fe1/IONP spheres made of silks blended at different ratios were obtained. Although the increased content of MS1Fe1 silk in particles resulted in an increased affinity of the spheres to IONPs, it decreased the binding of the composite particles to cancer cells. The H2.1MS1:MS1Fe1 particles prepared at a ratio of 8:2 and loaded with IONPs exhibited the ability to bind to the targeted cancer cells similar to the control spheres without IONPs. Moreover, when exposed to the alternating magnetic field, these particles generated 2.5 times higher heat. They caused an almost three times higher percentage of apoptosis in cancer cells than the control particles. The blending of silks enabled the generation of cancer-targeting spheres with a high affinity for iron oxide nanoparticles, which can be used for anti-cancer hyperthermia therapy.

Application of a three-dimensional (3D) breast cancer model to study macrophage polarization.

Knowledge of the tumor microenvironment is crucial for developing an effective strategy to treat cancer. Recently, anticancer therapies targeting macrophages have been intensively investigated. Increased understanding of the importance of the tumor microenvironment has led to the development of three-dimensional (3D) in vitro tumor models. However, established techniques for studying tumor-associated macrophages in vitro are limited. We have previously characterized a 3D breast cancer model consisting of breast cancer cells and fibroblasts cocultured on a silk scaffold. In the present study, the influence of this model on macrophage polarization was investigated. The expression of macrophage markers was studied using reverse transcription-quantitative PCR and flow cytometry. The activity of nitric oxide synthase and arginase in macrophages was also measured. The presented model appeared to induce the polarization of macrophages towards an M2 phenotype. In this 3D tumor model, the in vivo behavior of macrophages could be reproduced. This model may be beneficial for the study of tumor biology and for screening drugs.

MS1-type bioengineered spider silk nanoparticles do not exhibit toxicity in an in vivo mouse model.

Background: Due to factors such as silk sequence, purification, degradation, morphology and functionalization, each silk variant should be individually tested for potential toxicity. Aim:  In vivo toxicological evaluation of the previously characterized bioengineered H2.1MS1 spider silk particles that can deliver chemotherapeutics to human epidermal growth factor receptor 2-positive breast cancer. Materials & methods: Silk nanoparticles (H2.1MS1 and control MS1) were administered intravenously to mice, and then the organismal response was assessed. Several parameters of acute and subchronic toxicity were analyzed, including animal mortality and behavior, nanosphere biodistribution, and histopathological analysis of internal organs. Also, the complete blood count, as well as the concentration of biochemical parameters and cytokines in the serum, were examined. Results & conclusion: No toxicity of the systemically administrated silk nanosphere was observed, indicating their potential application in biomedicine.

Silk Particles as Carriers of Therapeutic Molecules for Cancer Treatment.

Although progress is observed in cancer treatment, this disease continues to be the second leading cause of death worldwide. The current understanding of cancer indicates that treating cancer should not be limited to killing cancer cells alone, but that the target is the complex tumor microenvironment (TME). The application of nanoparticle-based drug delivery systems (DDS) can not only target cancer cells and TME, but also simultaneously resolve the severe side effects of various cancer treatment approaches, leading to more effective, precise, and less invasive therapy. Nanoparticles based on proteins derived from silkworms' cocoons (like silk fibroin and sericins) and silk proteins from spiders (spidroins) are intensively explored not only in the oncology field. This natural-derived material offer biocompatibility, biodegradability, and simplicity of preparation methods. The protein-based material can be tailored for size, stability, drug loading/release kinetics, and functionalized with targeting ligands. This review summarizes the current status of drug delivery systems' development based on proteins derived from silk fibroin, sericins, and spidroins, which application is focused on systemic cancer treatment. The nanoparticles that deliver chemotherapeutics, nucleic acid-based therapeutics, natural-derived agents, therapeutic proteins or peptides, inorganic compounds, as well as photosensitive molecules, are introduced.

Implementation of a dynamic culture condition to the heterotypic 3D breast cancer model.

Cell culture system is used for a wide range of research and biotechnology production. Majority of in vitro cell studies are conducted as static, two dimensional (2D) dish culture system where cells grow in a monolayer. However, to better reflect the in vivo condition, three dimensional (3D) culture systems were introduced that allow investigating the cell-cell and cell-microenvironment interactions. In this work, the 3D breast cancer model was investigated. Previously, we developed a 3D breast cancer model that constituted of fibroblasts and breast cancer cells seeded on the silkworm silk scaffold. The dynamic culture condition that provides the medium flow and shear forces was implemented to the model. The dynamic conditions were compared to the static cultivation regarding its influence on the number of cells, their viability, scaffold penetration, and cells co-localization. The implication of the dynamic condition to the 3D cultures resulted in a higher number and viability of the cells compared with the static 3D cultures. In contrast to the static culture condition, during the dynamic cultivation cells penetrated entirely and evenly the inner parts of the scaffold. Moreover, in coculture, the transitions like a ratio of fibroblast to the cancer cells, fibroblast morphology, and their localization were similar in both types of culture conditions, but they proceeded much faster during the dynamic cultivation. The implementation of dynamic culture condition shortened the time needed to establish the settle 3D breast cancer model. The established dynamic cancer model can be used to study tumor biology and drug screening.