Interactions within the tumor microenvironment (TME) are complex and multidirectional, involving immune cells, fibroblasts, extracellular matrix components, exosomes, and numerous soluble mediators. Although the CRC microenvironment is challenging to study, it plays a key role in tumor progression and therapeutic response. Tumor-associated macrophages (TAMs) are particularly difficult to define, as they can shift between M1-and M2-like phenotypes depending on tumor context. Here, we used a direct co-culture of metastatic CRC cells with monocytes to assess how contact-dependent interactions affect the overall condition of the co-culture and monocyte activation. Cellular metabolic activity and viability were assessed, along with macrophage marker expression to determine the dominant phenotype (M1 or M2) in advanced CRC conditions. Additionally, levels of apoptosis and necrosis were evaluated, together with the expression of proteins involved in migration and angiogenesis. Our results demonstrate that direct co-culture represents a promising model for investigating tumor biology in the context of immune cell interactions and enables observation of key mechanisms underlying intercellular communication. Flow cytometric analysis revealed a significant increase in CD163 (67%) and CD206 (65%), suggesting the activation of M2-like features upon contact with advanced CRC. Furthermore, cancer cells cultured alone showed a greater tendency toward necrosis compared with co-culture conditions, indicating an association between the presence of immune cells and reduced tumor cell death. These findings highlight the strong modulation of the microenvironment by metastatic CRC and emphasize the importance of in vitro co-culture models as essential tools for studying tumor biology and intercellular communication mechanisms.

Small-cell lung cancer (SCLC) remains one of the deadliest cancers worldwide. Patients' survival remains poor due to its rapid growth, high metastatic rate and limited possibilities of treatment. For many years, SCLC management has been based mostly on chemo and radiotherapy. However, new therapeutic approaches have been proposed in the past few years, including immunotherapy, which is currently implemented in clinical practice. Unfortunately, in many cases, response to therapy, especially chemotherapy, remains poor, or the patient becomes resistant to initially effective treatment. One of the crucial problems during SCLC patient care is a lack of appropriate predictive biomarkers for various therapeutic approaches. Another critical issue is the scarcity of collected tissue during biopsy, which may be insufficient or of too poor quality for analysis. A liquid biopsy might be the key to solving both of those problems as it is collected in a non-invasive way and enables the measurement of various biomarkers, including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs). In this review, we discuss various approaches to potentially incorporating liquid biopsy into clinical application - as a companion to imaging during SCLC diagnostics, a new approach to molecular subtyping, and a material enabling predictive or prognostic biomarkers assessment. We also summarize ongoing clinical trials encompassing SCLC patients in which liquid biopsy is collected and examined.

Anti-proliferative and antioxidant activities of Pleurotus pulmonarius and P. citrinopileatuswater-soluble polysaccharides were examined using human colon adenocarcinoma (HT29) and human normal colonic epithelial (CCD841 CoTr) cell lines in vitro. Polysaccharides were prepared by cold and hot water extraction methods and chemically characterized. The monosaccharide analysis showed that all the preparations contained hexoses-mannose, glucose, galactose, and 3-O-methyl-hexose (identified as 3-O-methyl-galactose)-as well as trace amounts of pentose identified as arabinose. Linkage analysis, on the other hand, revealed a wide variety of monosaccharide linking bonds in the studied polysaccharides, depending on the polymer source and the method of isolation. Both fungal polysaccharide preparations with increasing concentrations showed lower DPPH free radical reducing activity. The degree of reduction depended on the origin of the compounds and the method of their preparation. Stimulation of nitric oxide (NOx) production by the tested cells was observed after their incubation with the polysaccharides. The tested compounds at lower concentrations (up to 500 mu g/mL) did not significantly influence the metabolic activity of the tumor and normal cells after 24 h of incubation. The viability of the tumor and normal cells was limited after the impact of the polysaccharides in the concentration range of 25-1000 mu g/mL. The hot and cold water isolated polysaccharides did not induce changes in the normal and tumor cell cytoskeleton organization. The paper presents newly prepared and characterized hot and cold isolated water-soluble polysaccharides with promising pro-health potential against tumor and normal human colon epithelium cells

Biomimetic catalysis using porphyrins enables gentle oxidation of terpenes with molecular oxygen and light. This study explores the photooxidation of (-)-myrtenol under visible light to synthesize new terpenoid products with promising biological activity. Among the porphyrins tested, tetraphenylporphyrin (H2TPP) exhibited the highest catalytic efficiency and stability in chloroform, producing myrtenal epoxide (ME) as the main product (with a molar conversion of myrtenol of 66.2 %), confirmed by NMR and MS analyses. Other substrates, i.e. perillyl alcohol and trans-pinocarveol, did not yield redox products. The antimicrobial activity of ME was assessed against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans using Disk Diffusion, Minimal Inhibitory Concentration, and Minimal Biofilm Eradication Concentration assays (using liquid ME) and the Quantitative Assay for Measuring the Antibiofilm Activity of Volatile Compounds (using volatile ME). Overall, ME displayed higher antimicrobial activity than myrtenol in the majority of the tests applied. The strongest effects were observed against C. albicans, followed by S. aureus, while the weakest activity was exhibited against Gram-negative bacteria. ME also showed cytotoxic effects on human colorectal cancer cells (HT-29) with significantly higher biological activity than that of (-)-myrtenol. Notably, ME at lower concentrations (5-50 µg/ml) promoted proliferation of normal cells while inhibiting the viability and proliferation of cancer cells. Porphyrin-based photooxidation is a sustainable method for converting biorenewable terpene feedstocks into new compounds that can be used in cancer treatment and antimicrobial therapy.

The modification of the surgical polypropylene mesh and the polytetrafluoroethylene vascular prosthesis with cecropin A (small peptide) and puromycin (aminonucleoside) yielded very stable preparations of modified biomaterials. The main emphasis was placed on analyses of their antimicrobial activity and potential immunomodulatory and non-cytotoxic properties towards the CCD841 CoTr model cell line. Cecropin A did not significantly affect the viability or proliferation of the CCD 841 CoTr cells, regardless of its soluble or immobilized form. In contrast, puromycin did not induce a significant decrease in the cell viability or proliferation in the immobilized form but significantly decreased cell viability and proliferation when administered in the soluble form. The covalent immobilization of these two molecules on the surface of biomaterials resulted in stable preparations that were able to inhibit the multiplication of Staphylococcus aureus and S. epidermidis strains. It was also found that the preparations induced the production of cytokines involved in antibacterial protection mechanisms and stimulated the immune response. The key regulator of this activity may be related to TLR4, a receptor recognizing bacterial LPS. In the present study, these factors were produced not only in the conditions of LPS stimulation but also in the absence of LPS, which indicates that cecropin A- and puromycin-modified biomaterials may upregulate pathways leading to humoral antibacterial immune response.

Colorectal cancer (CRC) is one of the most common and deadliest cancers worldwide. According to the GLOBOCAN (WHO) report in 2020, nearly 2 million patients were diagnosed globally. Despite the advances in cancer diagnosis and therapy, CRC remains a global challenge. Recently, attention has been paid to the tumor microenvironment (TME), which constitutes a significant part of the tumor and mainly includes various immune cells, fibroblasts, vascular cells, and extracellular elements, such as the extracellular matrix (ECM). Many components of the stroma initially exert an anti-tumor effect, but over time, they undergo functional transformation into elements that promote tumor growth. As a result, conditions conducive to further cancer development, invasion into local tissues, and distant metastasis arise. The microenvironment of colorectal cancer (CRC) may be an important direction in the search for therapeutic targets, but it requires further understanding. The main purpose of our review is to explain the role of the complex CRC microenvironment in the progression of this cancer and highlight the potential of targeted therapy directed at the TME. Therefore, continued research into its components and typical biomarkers is necessary to improve therapy and enhance the quality of life for patients.

In recent years, new sources of secondary metabolites (SMs) in medicinal plants have been identified, and the introduction of these plants into field conditions has been carried out to obtain chemically diverse standardized raw material (RM). An example is mountain arnica Arnica montana L., one of Europe’s endemic endangered medicinal plant species, commonly used in pharmacy, cosmetics, and medicine. Its inflorescences (Arnicae flos) are characterized by anti-inflammatory, antiradical, antioxidant, antibacterial, antifungal, and antitumor properties. The main goals of the present research included: (i) characterization of the chemical composition of the inflorescences of A. montana harvested in different development stages; and (ii) presentation of the role of the development stage and different extraction methods in the antitumor activity of extracts through analyses of apoptosis, autophagy, and necrosis induction in human cervical carcinoma HeLa, human colon carcinoma HT29, and human colon metastatic carcinoma SW620 cell lines. The development stage was found to modify the composition of pharmacologically active substances, e.g., sesquiterpene lactones (SLs), flavonoids (Fs), and essential oil (EO), in arnica inflorescences. The content of Fs and EO increased during flowering to the full flowering phase; however, the highest content of SLs was noted in the full flowering phase and at the end of flowering. More promising results, i.e., a relatively high level of apoptosis and a low level of necrosis induced by the arnica extracts, were demonstrated in the HeLa cell line (full flowering; concentration: 0.5 µL/mL), the HT29 cell line (beginning of flowering; concentration: 0.5 µL/mL), and the SW620 cell line (stage of yellow buds; concentration: 1 µL/mL). This extremely valuable medicinal plant species provides a very broad range of RMs (e.g., inflorescences, rhizomes, roots, achenes, and all plant); therefore, attention should be paid to the more frequent use of water as a solvent in studies on the biological activity of mountain arnica extracts.

Arnica montana L. is one of Europe's endemic endangered medicinal plants, with diverse biological activities commonly used in medicine, pharmacy, and cosmetics. Its flower heads are a rich source of raw material, with antibacterial, antifungal, antiseptic, anti-inflammatory, antiradical, antioxidant, and antitumor properties. The objective of the present study was (i) to characterize the chemical composition of flower heads of A. montana plants cultivated under nitrogen fertilization, (ii) to identify the impact of the nitrogen fertilization and extraction method (water, ethanol) on the antioxidant activity of extracts, and (iii) to determine the role of different nitrogen doses applied during plant cultivation and different extraction methods in the anticancer activity of the extracts through analysis of apoptosis and autophagy induction in HT29, HeLa, and SW620 cell lines. The present study shows that nitrogen is a crucial determinant of the chemical composition of arnica flower heads and the antioxidant and anticancer activity of the analyzed extracts. Nitrogen fertilization can modify the composition of pharmacologically active substances (sesquiterpene lactones, flavonoids, essential oil) in Arnicae flos. The content of sesquiterpene lactones, flavonoids, and essential oil increased with the increase in the nitrogen doses to 60 kg N ha(-1) by 0.66%, 1.45%, and 0.27%, respectively. A further increase in the nitrogen dose resulted in a decrease in the content of the analyzed secondary metabolites. Varied levels of nitrogen application can be regarded as a relevant way to modify the chemical composition of arnica flower heads and to increase the anticancer activity, which was confirmed by the increase in the level of apoptosis with the increase in fertilization to a level of 60 kg N ha(-1). The fertilization of arnica plants with low doses of nitrogen (30 and 60 kg N ha(-1)) significantly increased the LOX inhibition ability of the ethanol extracts. The present study is the first report on the anticancer activity of A. montana water extracts, with emphasis on the role of water as a solvent. In further studies of factors modifying the quality of Arnicae flos, attention should be paid to the simultaneous use of nitrogen and other microelements to achieve synergistic results and to the possibility of a more frequent use of water as a solvent in studies on the biological activity of A. montana extracts.

Collagen-sealed polyester (PET) prostheses are commonly used in reconstructive vascular surgery due to their self-sealing properties. To prevent post-surgical infection, different modification methods have been tested but so far none have showed long-term satisfactory efficiency. For this reason, in the present study, a commercial collagen-sealed PET prosthesis was coated by a highly adhesive poly (L-DOPA) layer maintaining the sealing protein without losing the original properties and functionality. This modified (as proven by SEM, FTIR, XPS and contact angle) graft exhibited comparable wettability and elasticity as pristine commercial graft, as well as reduced hemolysis-inducing effect, lowered toxicity against human endothelial cells and reduced toxicity in Danio rerio model. Poly (L-DOPA)-coated grafts were shown to bind six times more aminoglycoside antibiotic (gentamicin) than pristine graft. Poly (L-DOPA)-coated antibiotic-bound prostheses exhibited an improved antibacterial activity (bacterial growth inhibition and anti-adhesive capacity) in comparison with pristine antibiotic-bound graft. Overall, poly (L-DOPA)-coatings deposited on PET vascular grafts can effectively functionalize collagen-sealed prostheses without the loss of protein sealing layer and allow for antibiotics incorporation to provide higher safety in biomedical applications.

BACKGROUND:Colorectal cancer is one the most lethal cancers worldwide. Since chemotherapy is burdened with harmful effects, agents capable of enhancing the chemotherapeutic effect are being sought. Ursolic acid (UA) and oleanolic acid (OA) were analyzed for such properties. The aim of the study was to evaluate the ability of UA and OA administered individually and in combination with each other and/or a cytostatic drug camptothecin-11 (CPT-11) to limit the viability and migration of colorectal cancer cells.MATERIALS AND METHODS:The cytotoxic effect of UA, OA and CPT-11 and impact on normal and cancer cell migration rate were assessed. Furthermore, the effect on factors crucial in cancer metastasis: MMP-2 and -9, uPA/uPAR, and E-cadherin were assessed with ELISA, Western Blotting and immunofluorescence assays. Statistical analysis was performed with One-Way Anova with Dunnett's test.RESULTS:The studied compounds exhibited the most favorable properties, i.e. they reduced the viability and migration of cancer cells. Furthermore, the secretion, activity, and cellular level of cancer MMP-2 and -9 were decreased, as a result of uPA/uPAR down-regulation. The agents also increased the level of cellular E-cadherin. The effect of the studied agents on normal cells was milder.CONCLUSIONS:The compounds exhibited stronger activity when administered in combination and, combined with CPT-11, enhanced anti-tumorigenic activity of the drug. The migration-limiting activity was based on down-regulation of the uPA/uPAR-dependent MMP pathway. Moreover, UA and OA exhibited a protective effect towards normal cells.

The aim of this research was to determine the biological activity of (1→3),(1→4)-α-D-glucan from mango (Mangifera indica L.) fruits on human colon carcinoma cells HT29 (Duke's A) and SW620 (Duke's C).Many natural substances have been described to show anti-tumor activity or limit the dissemination of transformed cells.Among them, there are polysaccharides, which are biological macromolecules consisting of different amounts of monosaccharides linked by glycosidic bonds.One of such polymers is the water-soluble (1→3),(1→4)-α-D-glucan from mango fruits.The isolated (1→3),(1→4)-α-D-glucan did not induce a significant reduction in the number of tumor cells.The viability of cells did not drop below 94% upon the use of the polymer at the concentration of 225 µg/mL.The SW620 cells were more sensitive than HT29, showing a significant decrease in the cellular metabolism after incubation with the polysaccharide at a 250 µg/mL concentration.No significant changes in the morphology of both tumor cell types after the incubation with the (1→3),(1→4)-α-D-glucan were observed.The release of nitric oxide (NOx) by the tested cells was dependent on the polysaccharide concentration.There were differences in the NOx release profile depending on the type of cells.The (1→3),(1→4)-α-D-glucan decreased the release of IL-1β and IL-6 by both types of cells and increased the level of IL-10.Moreover, the α-D-glucan showed ferric reducing power and reduced the level of the DPPH free radical.Concluding, the analyzed (1→3),(1→4)-α-D-glucan isolated from mango fruits can limit the development of neoplastic cells not through direct cytotoxic action but via antioxidant and immunomodulatory activities.

Polyester (PET) prostheses are commonly used in reconstructive vascular surgery. The most serious complication after implantation is early or late infection of the graft. Therefore, there is high demand to protect prosthesis against bacterial adhesion and biofilm development. For this reason, in this work PET prostheses were first coated by highly adhesive polycatecholamine layer. The grafts were then coupled with gentamicin and studied in relation to morphological and structural properties, biological safety (contact with blood, reaction of vascular endothelial cells (HUVEC), Danio rerio fish), drug release and antibacterial activity. Among two tested catecholamine monomers, L-DOPA was found to be more effective precursor in this process than dopamine. For L-DOPA, assistance of Cu2+, Mg2+ and Na+ ions seems to increase the amount of further immobilized drug. Coated prostheses exhibited greater human endothelial cell proliferation increase and lower cytotoxic effect than uncoated. The modification reduced the hemolysis observed for pristine commercial graft and limited the rate of abnormalities in D. rerio larvae, confirming the safety of the proposed modification. The coating allowed to double the amount of immobilized antibiotic in comparison with uncoated graft which resulted in increased antibacterial activity and reduced bacterial adhesion against 4 bacterial strains prevalent in biomaterials infections. Overall, poly(L-DOPA)-coatings deposited on PET vascular grafts can effectively functionalize these prostheses for higher safety in biomedical applications.

The present study aimed to demonstrate Lentinus (formerly Pleurotus) sajor-caju (PSC) as a good source of pro-health substances. It has also shown that supplementation of its culture medium with cow milk may further improve its beneficial properties. Intracellular fractions from fungi grown on a medium supplemented with cow milk were analyzed using various biochemical methods for determination of the nutrient composition. Furthermore, anti-cancer properties of selected extracts were investigated on colorectal cancer cell lines (HT-29, LS 180, and SW948) in vitro. Biochemical analysis showed enrichment in health-enhancing compounds, such as proteins or polysaccharides (about 3.5- and 4.5-fold increase in concentration of proteins and carbohydratesin extracts of mycelia cultured on whole milk (PSC2-I), respectively), with a decrease in the level of free radicals (10-fold decrease in extract grown on milk and medium mixture (1:1) (PSC3-II)), which was related to increased catalase and superoxide dismutase activity (7.5-fold increase in catalase activity and 5-fold in SOD activity in PSC3-II compared to the control). Moreover, the viability of the cancer cells was diminished (to 60.0 ± 6.8% and 40.0 ± 8.6% of the control, on HT-29 and SW948 cells, respectively), along with pro-apoptotic (to 18.8 ± 11.8 and 14.7 ± 8.0% towards LS 180 and SW948 cells, respectively) and NO-secreting effects (about 2-fold increase) of the extracts. This study suggests that PSC has multiple nutritional and anti-cancer properties and can be used as a source of healthy biomolecules in modern medicine or functional foods.

In this study, the influence of two subfractions (with previously proven anti-cancer properties) isolated from wood rot fungus Cerrena unicolor on the formation of a fibrin clot was investigated in the context of potential use as fibrin glue and sealant enhancers and potential wound healing agents. With the use of ROTEM thromboelastometry, we demonstrated that, in the presence of fibrinogen and thrombin, the S6 fraction accelerated the formation of a fibrin clot, had a positive effect on its elasticity modulus, and enhanced the degree of fibrin cross-linking. The S5 fraction alone showed no influence on the fibrin coagulation process; however, in the presence of fibrin, it exhibited a decrease in anti-proliferative properties against the HT-29 line, while it increased the proliferation of cells in general at a concentration of 100 µg/mL. Both fractions retained their proapoptotic properties to a lesser degree. In combination with the S6 fraction in the ratio of 1:1 and 1:3, the fractions contributed to increased inhibition of the activity of matrix metalloproteinases (MMPs). This may suggest anti-metastatic activity of the combined fractions. In conclusion, the potential of the fractions isolated from the C. unicolor secretome to be used as a means of improving the wound healing process was presented. The potential for delivering agents with cytostatic properties introduced far from the site of action or exerting a pro-proliferative effect at the wound site with the aid of a fibrin sealant was demonstrated.

The biochemical properties and anti-tumorigenic activity of Cerrena unicolor (CU) and Pycnoporus sanguineus (PS) towards colon cancer cells and the effect of supplementation of the fungal culture medium with cow milk on these activities were examined. CU1-II and PS4-II exhibited anticancer properties through various mechanisms. The extracts at the 200 µg/mL concentration significantly decreased the viability of HT-29 and SW948 cells. They also exhibited pro-apoptotic properties towards the cancer cell lines (HT-29, LS 180, and SW948). Furthermore, culturing the studied fungi on milk-supplemented media may improve the pro-health properties of both milk and mushrooms. The extracts had a higher concentration of proteins, lower levels of free amino acids, and higher content of phenolic compounds than milk. They also exerted a free radical scavenging effect, which may be connected with the high activity of catalase and superoxide dismutase. The tested extracts exhibited anticancer activity: C. unicolor grown on the medium without milk and P. sanguineus grown on the medium with milk. The CU1-II and PS4-II extracts exhibited the strongest anticancer properties; however, PS4-II exerted a milder effect on normal CCD 841 CoTr cells than CU1-II. CU3-II exerted the mildest effect among all extracts on both normal and cancer cells.

Various phytochemical studies have revealed that jackfruit (Artocarpus heterophyllus Lam.) is rich in bioactive compounds, including carotenoids, flavonoids, volatile acids, tannins, and lectins. The aim of the study was to analyze the biological activity of water-soluble polysaccharide (WSP) isolated from jackfruit and to assess its immunomodulatory, cytotoxic, and anti-oxidative effects on human colon carcinoma cells in vitro. The neutral red (NR) uptake assay revealed no toxic influence of the polymer on the viability of tumor cells (HT29 and SW620). After 24 h and 48 h of incubation, the cellular viability was not lower than 94%. The metabolic activity of the cells (MTT) at the compound concentration of 250 µg/mL was higher than 92% in comparison to the control. WSP (250 µg/mL) exerted no significant effect on the morphology of the cells was determined by May-Grünwald-Giemsa staining. WSP changed nitric oxide (NOx) production by the tumor cells depending on the time of incubation and prior 2-h stimulation of the cells with E. coli 0111:B4 LPS. It significantly stimulated IL-1β production by the tumor cells. The IL-6 level increased but that of IL-10 decreased by a WSP concentration-dependent manner. No such effect was detected in SW620. The WSP had antioxidant properties. In conclusion, water-soluble polysaccharide isolated from A. heterophyllus exhibits significant biological activity towards many types of both normal and cancerous cells. Therefore, it may be considered as a useful agent in the protection of human health or in functional and dietary nutrition.

Background: Cancer is one of the main causes of death worldwide. Contemporary therapies, including chemo- and radiotherapy, are burdened with severe side effects. Thus, there exists an urgent need to develop therapies that would be less devastating to the patient’s body. Such novel approaches can be based on the anti-tumorigenic activity of particular compounds or may involve sensitizing cells to chemotherapy and radiotherapy or reducing the side-effects of regular treatment. Objective: Natural-derived compounds are becoming more and more popular in cancer research. Examples of such substances are Ursolic Acid (UA) and Oleanolic Acid (OA), plant-derived pentacyclic triterpenoids which possess numerous beneficial properties, including anti-tumorigenic activity. Results: In recent years, ursolic and oleanolic acids have been demonstrated to exert a range of anticancer effects on various types of tumors. These compounds inhibit the viability and proliferation of cancer cells, prevent their migration and metastasis and induce their apoptosis. Both in vitro and in vivo studies indicate that UA and OA are promising anti-cancer agents that can prevent carcinogenesis at each step. Furthermore, cancers at all stages are susceptible to the activity of these compounds. Neoplasms that are formed in the gastrointestinal tract, i.e. gastric, colorectal, pancreatic, and liver cancers, are among the most common and most lethal malignancies. Their localization in the digestive system, however, facilitates the action of orally-administered (potential) anti-cancer agents, making chemopreventive drugs more accessible. In this paper, the anti-tumorigenic effect of ursolic and oleanolic acids on gastric, colon, pancreatic, and liver cancers, as well as the mechanisms underlying this process, are presented.

The aim of this study was to investigate the anticancer and antioxidant activities of low molecular weight subfractions isolated from secondary metabolites produced by the wood degrading fungus Cerrena unicolor . Human colon cancer cells (stage I) HT-29 and human normal colon epithelial cells CCD 841 CoTr were used in the research. The present study demonstrated that the low molecular weight subfractions exhibited inhibitory activity towards human colon cancer cells HT-29 at a concentration range of 25–200 μg/mL. All 6 subfractions inhibited proliferation of cells down to 47.5–9.2% at the highest concentrations in a dose-dependent manner. The most desired activity was exhibited by subfractions S, 3, 4, and 5, as the proliferation of HT-29 cells was inhibited to the greatest extent (16.5, 47.5, 42.7, and 26.1% of the control, respectively), while the effect on CCD 841 CoTr cells was the mildest (inhibition to 54.4, 71.4, 79.4, and 53.4%, compared to the control, respectively). The microscopic observation revealed that all extracts induced programmed cell death, i.e. apoptosis (up to 44.4% (subfraction 6) towards HT-29 and less than 20% (most fractions) towards CCD 841 CoTr), with no or a significantly low level of necrosis in both cell lines at the same time.