Enhanced NK cell proliferation by stress-induced feeder cells

Enhanced NK cell proliferation by stress-induced feeder cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhanced NK cell proliferation by stress-induced feeder cells Donghyun Lee, Myeongkwan Song, Soonjo Kwon This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3918793/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Natural Killer (NK) cells, integral to the innate immune system, are notable in cell therapies because for their applicability in allogeneic treatments, distinguishing them from T cells typically employed in conventional cell therapies. However, their limited half-life poses a challenge for therapy. Although attempts to leverage feeder cells are common, safer methods are needed to mitigate the associated risks. In our study, an upregulation in the expression of 4-1BBL in Colo-205 cells under extracellular stresses such as hypoxia and cytochalasin D was observed. This enhanced binding to the 4-1BB receptors on NK cells promotes proliferation in NK cells. Elevated CD56 expression of a marker strongly linked to NK cell proliferation in co-culture further supports this process. Applying extracellular stressors, specifically hypoxia and cytochalasin D, to Colo-205 cells successfully tailored feeder cells, significantly enhancing NK cell proliferation. NK cell proliferation Colo-205 feeder cell 4-1BB/4-1BBL binding CD56 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Natural Killer (NK) cells are prototypic members of the innate lymphoid cell (ILC) family, and in humans, NK cells are phenotypically characterized by being CD3 − and CD56 + [ 1 , 2 ]. They are predominantly found in the peripheral blood, constituting 5–15% of peripheral blood lymphocytes. According to the missing-self hypothesis, NK cells identify target cells by detecting the presence or absence of major histocompatibility complex (MHC) class I molecules [ 3 ]. In contrast, T cells distinguish between self and non-self based on the structure of MHC class I, initiating an attack on the target cell if it is identified as non-self. Essentially, this limitation limits the application of T-cell therapy to autologous applications. NK cells, unlike T cells, are not restricted by MHC class I molecules during target recognition, making them suitable for allogeneic cell therapy [ 4 ]. These advantages render NK cells compelling candidates for off-the-shelf cellular therapeutics. Consequently, recent efforts have focused on substituting NK cells with T cells as a source of cellular therapeutics. However, a significant drawback associated with NK cells is their limited half-life compared to other immune cells, which poses a substantial challenge in the proliferation in an in vitro setting [ 5 ]. To address the limitations of NK cells, various approaches are underway, including gene editing [ 6 – 8 ] and culturing in bioreactors [ 9 , 10 ]. Among these methods, co-culture with feeder cells is the most effective strategy for generating NK cells in vitro. This approach has been used to enhance NK cell proliferation. The proliferation NK cell relies on cell interactions where 4-1BB on NK cells binds to 4-1BBL on feeder cells [ 11 ]. The binding of 4-1BB/4-1BBL represents a cell-cell interaction that triggers the upregulation of NF-κB, ATF2, and c-Jun expression. This initiates a cascade of events that promotes NK cell proliferation, as evidenced in previous studies [ 7 , 8 , 11 , 12 ]. Most studies have achieved this via gene editing of feeder cells, a method associated with the drawback of potential induction of mutations during the process. Therefore, safer alternatives should be explored. Consistent with numerous previous studies, the interaction between 4-1BB and 4-1BBL resulted in the upregulation of CD56 (neural cell adhesion molecule, NCAM-1) expression in NK cells. Notably, CD56 bright NK cells, characterized by increased CD56 expression, exhibit more vigorous proliferation, as highlighted in previous studies [ 13 – 15 ]. While the mechanism underlying CD56’s promotion of NK cell proliferation remains unclear, some researchers suggest that increased cytokine release as a potential contributing factor, corresponding with the upregulated expression of CD56 [ 2 , 16 , 17 ]. In this study, Colo-205 cells were selected as the preferred feeder cells among various candidates. Colo-205 tumor cells are known for their remarkably high levels of 4-1BBL expression [ 18 ]. Modifications were implemented to inhibit feeder cell proliferation without resorting to gene editing, involving pretreatment with cytochalasin D under hypoxic conditions. Furthermore, subjecting Colo-205 cells to extracellular stress during pretreatment resulted in the upregulation of 4-1BBL expression. Subsequently, we measured cell expansion to validate the enhanced proliferation of NK cells during coculture. Furthermore, we examined the phenotypic changes in NK cells. At this point, we divided the culture process into static and dynamic conditions to evaluate the feasibility of employing feeder cells in dynamic environments, such as bioreactors. Our findings revealed that pre-treated and modified Colo-205 cells, serving as feeder cells, significantly enhanced NK proliferation under both static and dynamic conditions. MATERIALS AND METHODS Cell culture Colo-205 human colon cancer cells were purchased from the Korea Cell Line Bank (KCLB Korea), and KHYG-1 human natural killer cells were purchased from AcceGen Biotech (ABC-TC0506, USA). Colo-205 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin at 37°C in a humidified CO 2 incubator with 5% CO 2 . KHYG-1 cells were cultured under identical conditions as Colo-205, supplemented with an additional 100 U/mL of recombinant human interleukin-2 (Peprotech, 200-02). Both cell types were seeded in a 90 × 20 mm cell culture dish (SPL, 20101). The medium was changed every 3–4 days. Feeder cell modification To induce hypoxic conditions in Colo-205 cells for modification, sodium sulfite and cobalt chloride were utilized, while cytochalasin D was employed to enhance the efficiency of feeder cell utilization. Colo-205 cells were seeded at a density of 5 × 10 4 cells/cm 2 in a 60 × 15 mm cell culture dish (SPL, 20060). Subsequently, the culture medium was replaced with 3 mL of fresh media supplemented with 4 mM sodium sulfite (Sigma), 100 µM cobalt chloride (Sigma), and 3 µM cytochalasin D (Cell Signaling Technology). Treated Colo-205 cells were then cultured for 24 h at 37°C in a humidified incubator with 5% CO 2 . Exosome isolation methods To analyze the inhibitory effect of cytochalasin D on exosomes, exosomes were isolated from cytochalasin-D-treated Colo-205 cells using a centrifugation-based method. Initially, the culture media of Colo-205 cells were collected and subjected to centrifugation at 2000 × g and 4°C twice. The supernatant obtained from this process was further subjected to ultracentrifugation at 100,000 × g and 4°C, following which the pellet containing exosomes was collected. Next, nanoparticle tracking analysis (NTA) was conducted using the ZetaView® PMX-220 (Particle Metrix GmbH, Germany) to quantify the concentration of exosomes released by Colo-205 cells. Co-culture of NK cells and feeder cells A T-75 flask (SPL 70075) was utilized for the co-culture of NK cells and feeder cells. Under dynamic conditions, the co-culture was carried out on a digital rocker RK-2D (DAIHAN, Korea) to subject the NK cells to shear stress. The RPM speed of the digital rocker was set to 10 RPM. The seeding cell number ratio between NK cells and feeder cells was set at 100:1. The culture period extended for four days. Additionally, on the second day of co-culture, the medium was changed to prevent NK cell proliferation caused by media depletion. Gene expression profiling by RT-qPCR RT-qPCR was performed to determine gene expression. The proliferation markers Ki-67 ( MKI67 ), proliferating cell nuclear antigen ( PCNA ), and minichromosome maintenance complex component 2 ( MCM2 ), were chosen as gene indicators for cell proliferation. Stress-related genes in the feeder cells included those encoding for MHC class I polypeptide-related sequence A ( MICA ), MHC class I polypeptide-related sequence B ( MICB ), and UL16 binding protein 1 ( ULBP1 ). Genes encoding for tumor necrosis factor superfamily member 9 ( TNFSF9 ) and tumor necrosis factor receptor superfamily member 9 ( TNFRSF9 ) were selected as genes related to 4-1BBL and 4-1BB in feeder and NK cells. The CD56 gene was chosen to indicate the expression of its corresponding protein in NK cells. IFN-γ , TNF-α , and IL-10 were chosen as genes representing cytokines in NK cells, with glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) serving as the housekeeping gene (Table 1 ). To isolate total cellular mRNA from K562 cells, the cells were treated with the TRIzol reagent in cell plates for 5 min. Trizol reagent and cellular mRNA were then transferred to microcentrifuge tubes, and 200 µL of chloroform was added, followed by centrifugation at 12,000 × g and 4 ℃ for 10 min. The supernatant was then transferred to a new microcentrifuge tube. To the tube, an equal volume of isopropanol was added, followed by additional centrifugation at 12,000 × g and 4 ℃ for 5 min. The supernatant was then discarded, and 75% ethanol was added to the tube before further centrifugation at 12,000 × g and 4 ℃ for 5 min. The supernatant was removed and air-dried. To dissolve the mRNA pellet, RNase-free water was added, and the mixture was incubated at 60 ℃ for 10 min. The mRNA concentration was quantified using a Nanodrop DS-11 (DeNovix, Wilmington, DE, USA). To synthesize cDNA from mRNA (1 µg), we used a cDNA synthesis kit (Takara) according to the manufacturer’s protocol. The synthesized cDNA was mixed with TB Green® Premix Ex Taq™ II (Takara), the appropriate target gene primer, and RNase-free water, after which RT-qPCR was performed. The primer sequences are listed in Table 1 . Fold changes in target gene expression levels were calculated using the 2 −ΔΔCt method. Table 1 Primer sequences Target gene Sequence Size GAPDH F : 5’-GAAATCCCATCACCATCTTCCAGG-3’ R : 5’-GAGCCCCAGCCTTCTCCATG-3’ 120 MKI67 F : 5’-CGTCCCAGTGGAAGAGTTGT-3’ R : 5’-CGACCCCGCTCCTTTTGATA-3’ 143 PCNA F : 5’-CGGTTACTGAGGGCGAGAAG-3’ R : 5’-GCTGAGACTTGCGTAAGGGA-3’ 94 MCM2 F : 5’-ATCGTGGTACTGCTATGGCG-3’ R : 5’-TGGAGGTGAGGGCATCAGTA-3’ 135 MICA F : 5’-CTAAAGTCTGAGAGAGGGAAGTCG-3’ R : 5’-CGAAGACTGTGGGGCTCTTATAC-3’ 71 MICB F : 5’-TAAAGTCTGCGAGGAGGAAGTCG-3’ R : 5’-GACTGCACAGATCCATCCTGG-3’ 92 ULBP1 F : 5’-GACAGCACTTCATCCTGGAGC-3’ R : 5’-CAGAGAGGGTGGTTTTGTTGGA-3’ 166 TNFSF9 F : 5’-CCAGGCTAGGGGGCTATAGAA-3’ R : 5’-GAGGCTGGGGATGAGTCTTT-3’ 96 TNFRSF9 F : 5’-CGTTGCTCTTCCTGCTGTTC-3’ R : 5’-CTTCTTCTGGAAATCGGCAGC-3’ 153 CD56 F : 5’-CACTTATCGCTGTGAGGGCA-3’ R : 5’-TTTGTCCAGCTCATGGTGGG-3’ 192 IFN-γ F : 5’-GAGTGTGGAGACCATCAAGGAAG-3’ R : 5’-TGCTTTGCGTTGGACATTCAAGTC-3’ 124 TNF-α F : 5’-CGAGTGACAAGCCTGTAGCC-3’ R : 5’-GGACCTGGGAGTAGATGAGGT-3’ 165 IL-10 F : 5’-GCAAAACCAAACCACAAGACAG-3’ R : 5’-TCAGGAGGACCAGGCAACAG-3’ 78 Protein expression profile study by flow cytometry To assess CD56 expression, flow cytometry was conducted following the manufacturer’s protocol of the APC-conjugated anti-human CD56 antibody (Cell Signaling Technology). Post co-culturing feeder cells and NK cells, washing was carried out using 0.5% BSA (Sigma) DPBS buffer at 182 × g and 4 ℃ for 3 min. Following this, the APC-conjugated anti-human CD56 antibody was diluted at a ratio of 20:1 in 0.5% BSA DPBS and added to the NK cells, followed by incubation on ice for 60 min. The cells were subsequently washed twice with 0.5% BSA DPBS before flow cytometry analysis. Stained cells were analyzed using a BD® LSR II Flow Cytometer (BD Biosciences, USA) employing the FACSDiva v9.0 software (BD Biosciences, USA). Data analysis was performed using the FlowJo™ v10.8 software (BD Biosciences, USA). Statistical analysis Collected data are presented as mean ± SEM (standard error of the mean (SEM). Differences between the control and experimental groups were assessed using the Student’s t-test. Differences were considered statistically significant at P < 0.05. GraphPad Prism v10.1.2 software (GraphPad, USA) was used for statistical analysis. RESULTS AND DISCUSSION Inhibition of feeder cell proliferation by stress factors To improve the effectiveness of Colo-205 cells as feeder cells, they underwent a 24 h treatment with cytochalasin D under hypoxic conditions, followed by three days of culturing under normal conditions. Subsequently, viable cell density was assessed using a trypan blue assay (Fig. 2 A). All groups, including the hypoxia-alone, cytochalasin D-alone, and combined hypoxia and cytochalasin D groups, exhibited lower cell densities compared to the control. Each group demonstrated viable cell densities of approximately 89.46%, 48.52%, and 35.58%, respectively, compared with the control. The results were considered statistically significant. Additionally, we aimed to evaluate the impact of these stress factors on Colo-205 cell proliferation by examining marker genes associated with proliferation through RT-qPCR. The selected markers included MKI67 , which indicates ongoing cell cycle progression [ 19 , 20 ], PCNA , involved in forming a protein complex crucial for DNA replication during the DNA synthesis phase [ 20 , 21 ], and MCM2 , which participates in DNA replication initiation by interacting with the origin recognition complex (ORC) [ 20 , 22 ], the initiator of DNA replication. RT-qPCR analysis was conducted for these markers (Fig. 2 B). The experiment yielded conclusive results, indicating a statistically significant downregulation in the expression of all three genes compared to that in the control across all groups. In the combined hypoxia and cytochalasin D group, MKI67 expression decreased by 0.33-fold, PCNA by 0.52-fold, and MCM2 by 0.10-fold compared to the control group. These RT-qPCR results regarding proliferation-related markers corroborated the findings from the trypan blue assay described earlier. The results of the two experiments showed consistent tendencies. Specifically, it was evident that cell density was significantly decreased and gene expression was downregulated, particularly in the groups treated with cytochalasin D. The variations in results between groups treated with and without cytochalasin D can be attributed to the distinct mechanisms through which each stress factor induces cell cycle arrest. HIF-1α accumulates within cells under hypoxic conditions, is known to regulate the p27 expression, thereby preventing cells in the G1 phase from entering the S phase [ 23 ]. However, under normoxic conditions, the accumulated HIF-1α is decomposed, allowing the cell cycle to proceed normally again [ 24 ]. Therefore, the decrease in cell density and expression of proliferation-related markers in the hypoxia-only treatment group was less pronounced than in the other groups. Cytochalasin D impedes cell cycle progression by inhibiting actin polymerization within cells [ 25 , 26 ]. This action primarily affects the M phase, necessitating actin polymerization and inhibiting the cell membrane formation [ 27 ]. Unlike the accumulation of HIF-1α under hypoxic conditions, the effects of cytochalasin D persisted even under normal conditions. Consequently, cell density and the expression of proliferation-related genes remained consistently low, particularly in the groups treated with cytochalasin D. Modulation of NKG2DL expression in feeder cells induced by stress factor The expression of NKG2DL, such as MICA/B and ULBP1-6, is upregulated in abnormal cells, such as cancer cells [ 28 ]. Additionally, the expression is also upregulated when extracellular stress is applied to these abnormal cells [ 29 , 30 ]. When NKG2DL binds to the NKG2D receptor on the surface of NK cells, NKG2D is activated. Activated NKG2D plays a crucial role in promoting cell proliferation through various signaling pathways [ 31 ]. To confirm alterations in the expression of stress-related ligands in feeder cells under stress conditions such as hypoxia, cytochalasin D, MICA , MICB , and ULBP1 as marker genes for stress-related marker genes, and RT-qPCR was conducted (Fig. 3 ). The experiment confirmed that the expression was upregulated in all groups compared to that in the control group. Especially in the group subjected to both hypoxia and cytochalasin D group, the MICA exhibited a 4.04-fold increase, MICB showed a 2.94-fold increase, and ULBP1 demonstrated a 1.61-fold increase, signifying a substantial elevation due to the other stress factors employed in this study. Additionally, the experimental results were statistically significant for all outcomes except ULBP1 in the hypoxia-alone group. In addition to NK cell activation through 4-1BB/4-1BBL binding mentioned in the introduction we anticipate the promotion of NK cell proliferation through the signaling pathway mediated by NKG2D/NKG2DL binding between NK cells and feeder cells. Inhibition of exosome release in feeder cells mediated by stress factors Under hypoxic conditions, the release of exosomes by tumor cells is higher than that under normal conditions [ 32 , 33 ]. Furthermore, tumor exosomes released under hypoxic conditions actively inhibit NK cell function via NKG2DL on the exosome surface [ 34 – 37 ]. Milad Moloudizargari et al. reported that NKG2D, which normally binds to MICA/B on the tumor cell surface, binds to MICA on the surface of tumor exosomes. This enables tumor cells to evade NK-mediated antitumor immunity through exosomal MICA/B shedding, a phenomenon termed immune escape [ 34 ]. Guy Berchem et al. reported that tumor exosomes inhibit the function of NKG2D via the expression of TGF-β [ 35 ]. Hence, to prevent the inhibition of NK cell function by tumor exosomes, it is imperative to suppress exosome release from tumor cells for use as feeder cells. Cytochalasin D is known to inhibit actin polymerization and is known for its ability to block exosome release [ 38 ]. Thus, to maintain optimal NK cell function, we aimed to create feeder cells with suppressed exosome release after cytochalasin D treatment. Therefore, to determine the effect of cytochalasin D treatment on exosome release from feeder cells, we isolated exosomes from feeder cells by ultracentrifugation and analyzed them by NTA (Fig. 4 ). When the quantified data were analyzed, the exosome concentrations for each group were as follows: Control, 1.2 × 10 10 particles/mL; hypoxia-alone group, 2.0 × 10 10 particles/mL; cytochalasin D-alone group, 1.9 × 10 9 particles/mL; and the combined hypoxia and cytochalasin D group, 1.4 × 10 9 particles/mL. Compared with the control, the exosome release rates were approximately 166.67%, 15.83%, and 11.67%, respectively. Specifically, the group treated with hypoxia alone exhibited a 1.67-fold higher exosome concentration than the control. This result aligns with the concept that hypoxia induces tumor cells to release more exosomes, as explained earlier. Furthermore, the two groups treated with cytochalasin D exhibited comparable exosome concentrations. This similarity arises from the fact that actin polymerization was already inhibited by cytochalasin D in these groups, leading to the inhibition of exosome release regardless of the application of hypoxia. Enhanced 4-1BBL expression in feeder cells induced by stress factors Various tumor cells express 4-1BBL. According to Helmut R. Salih et al., Colo-205 cells exhibit the highest levels of 4-1BBL expression [ 18 ]. To evaluate the impact of stress factors on the expression of 4-1BBL in feeder cells, TNFSF9 (4-1BBL) was selected as a marker, and RT-qPCR was conducted (Fig. 5 ). All groups exhibited a statistically significant upregulation of TNFSF9 expression compared to the control, with specific fold changes as follows: 1.70-fold in the hypoxia-only group, 3.52-fold in the cytochalasin D-only group, and 4.15-fold in the combined hypoxia and cytochalasin D group compared to the control. While the correlation between extracellular stress and the expression of 4-1BBL has not been established, Lu Qiu et al. reported that cells subjected to oxidative stress exhibit increased 4-1BBL gene transcription. This effect is mediated by the intracellular antioxidant transcription factor NFE2L1, which promotes 4-1BBL gene transcription via ARE in the promoter region [ 39 ]. Feeder cells, following this modification, were co-cultured with NK cells to induce 4-1BB/4-1BBL binding. Subsequently, RT-qPCR was performed using TNFRSF9 (4-1BB) as the selected marker (Supplementary Fig. 1). In summary, the co-culture group under dynamic conditions exhibited a more pronounced upregulation of TNFRSF9 (4-1BB) expression than the co-culture group under static conditions. Additionally, TNFRSF9 (4-1BB) expression increased in the control group under dynamic conditions even in the absence of 4-1BB/4-1BBL binding. These results support the notion that regulation of 4-1BB expression in NK cells is influenced not only by 4-1BB/4-1BBL binding but also by the prevailing culture conditions. Enhanced proliferation of NK cells following co-culture with feeder cells We evaluated the extent to which NK cell proliferation enhancement by co-culturing the feeder cells previously subjected to modifications with NK cells. The experiments were categorized into three groups: NK cells cultured independently without feeder cells (control), NK cells co-cultured with unstressed feeder cells (non-stressed feeder cells), and NK cells co-cultured with stress-induced feeder cells (stressed feeder cells). To assess distinctions in the culture methods for each group, the cultures were segregated into static and dynamic conditions, with a total culture period of four days. Initially, we performed a trypan blue assay on NK cells after co-culturing with feeder cells to assess the extent of cell number expansion facilitated by the feeder cells (Fig. 6 A). Under dynamic conditions, all groups exhibited over a 2-fold increase in cell expansion compared to the control under static conditions (approximately 2.17-fold, 2.18-fold, and 2.34-fold), with all results reaching statistical significance compared to the control. Conversely, NK cells co-cultured with non-stressed feeder cells under static conditions displayed a cell number comparable to that of control cells (approximately 0.96-fold). However, NK cells co-cultured with stressed feeder cells under static conditions exhibited statistically significant cell expansion compared with the control (approximately 1.41-fold). This results indicate a significant increase in cell number compared with NK cells co-cultured with non-stressed feeder cells under static conditions. Subsequently, to assess the impact of co-culture with feeder cells on NK cell proliferation, we conducted RT-qPCR using the proliferation-related genes MKI67 , PCNA , and MCM2 as markers [ 20 ]. RT-qPCR was conducted using samples collected on days 2 and 4 of the incubation period (Fig. 6 B-D). On Day 2, the expression of the MKI67 gene was significantly upregulated in all groups, except for NK cells with non-stressed feeder cells in static conditions, compared to their respective controls (Fig. 6 B). Furthermore, the expression on Day 4 was significantly upregulated compared to the respective controls in all groups, except for the two groups of NK cells with non-stressed feeder cells under static and dynamic conditions. The disparity in MKI67 expression in NK cells resulting from feeder cell exposure to stress factors was notably and statistically significantly upregulated in NK cells with stressed feeder cells under static conditions on both days 2 and 4. On day 2, the expression of the PCNA gene showed significant upregulation in all groups, except for NK cells co-cultured with stressed and non-stressed feeder cells under static conditions, compared to their respective controls (Fig. 6 C). Furthermore, the expression on day 4 was significantly upregulated compared to the respective controls in all groups. The disparity in PCNA expression in NK cells resulting from feeder cell exposure to stress factors was notably and statistically significantly increased in NK cells with stressed feeder cells under static conditions on both days 2 and 4. Moreover, proliferation-related gene expression was downregulated in most cases in NK cells with and without feeder cells under dynamic conditions. A notable exception was found in the expression of PCNA in NK cells with non-stressed feeder cells under dynamic conditions, exhibiting a slight upregulation on day 4. Given that PCNA is involved in DNA damage repair, this upregulation suggests a potential response to repair the damage incurred by NK cells owing to their interaction with non-stressed feeder cells [ 21 ]. At the end of the Day 2 incubation period, the expression of the MCM2 gene was significantly upregulated in all groups, except for NK cells with non-stressed feeder cells under static conditions, compared to their respective controls (Fig. 6 D). Furthermore, the expression on day 4 was significantly upregulated compared to the respective controls in all groups. The significant upregulation of MCM2 expression in NK cells co-cultured with stressed feeder cells under static conditions on both days 2 and 4. When comparing the results, no statistically significant differences were observed between NK cells with and without feeder cells in dynamic conditions (Fig. 6 A). The proliferative enhancement effect induced by dynamic conditions on day 4 was deemed more significant comparatively to the impact of feeder cell presence, explaining the observed attribution. Huixun Du et al. reported two pathways for the enhancement of proliferation by externally applied shear stress: 1) Inhibition of the Hippo pathway, and 2) activation of transient receptor potential vanilloid type 4 (TRPV4) and piezo-type mechanosensitive ion channel component 1 (PIEZO1) [ 40 ]. Under shear stress conditions, inhibition of the Hippo pathway occurs, facilitating the translocation of phosphorylated Yes-associated protein (YAP) and PDZ-binding motif (TAZ) into the nucleus, thereby promoting the proliferation of immune cells [ 40 ]. Kevin P. Meng et al. reported that the activation of YAP inhibits T-cell activation and proliferation, supporting the enhancement mentioned earlier, of proliferation through the inhibition of the Hippo pathway [ 41 ]. Subsequently, findings, it was observed that the application of shear stress to cells activates the cation channels TRPV4 and PIEZO1. This activation leads to an augmented influx of Ca 2+ , and the heightened Ca 2+ influx, in turn, facilitates cell proliferation by regulating transcription factors [ 40 ]. Comparing the results on day 2, there was a more pronounced upregulation when cells were cultured under dynamic conditions compared to static conditions the expression of genes associated with proliferation exhibited (Fig. 6 B-D). These results further corroborate that the impact of dynamic conditions on proliferation was relatively more substantial than the influence of the presence of feeder cells. CD56 expression of NK cells following co-culture with feeder cells Upon engaging in 4-1BB/4-1BBL binding, NK cells undergo activation, characterized by elevated levels of CD56 (NCAM-1) expression [ 2 , 13 , 42 ]. To investigate changes in CD56 expression, we co-cultured NK cells with feeder cells previously subjected to modification, examining alterations at both the gene and protein levels in NK cells. The co-culture was performed according to a previously described procedure. RT-qPCR analyses were conducted using samples collected on days 2 and 4 of the culture period, with emphasis placed on the CD56 gene selected as the marker. Flow cytometry analysis was conducted on day 4, marking the completion of the culture. The anti-CD56 antibody (APC conjugate) was selected as a marker for this analysis (Fig. 7 A-B). RT-qPCR analysis confirmed a statistically significant upregulation in expression levels in all groups, regardless of the culture method, exceeding that of the respective controls (Fig. 7 A). Moreover, a notable increase in CD56 expression in NK cells was observed upon the exposure of feeder cells to stress factors. Specifically, this upregulation was notable in NK cells with stressed feeder cells under static conditions on day 2 and in NK cells with stressed feeder cells under dynamic conditions on day 4. On Day 2, CD56 expression in the dynamic condition group exhibited more pronounced upregulation within each group. This observation suggests that the expression of CD56 is notably influenced by culture conditions, similar to the previously depicted impact on proliferation. Feng Wang et al. reported an upregulation in the expression of cell adhesion molecule (CAM) proteins such as VCAM-1 and ICAM-1, similar to CD56, in human umbilical vein endothelial cells (HUVEC) subjected to shear stress [ 43 ]. Furthermore, Nina Schwankhaus et al. reported upregulation of ICAM-1 expression when SK-N-SH and LAN1 cells were exposed to shear stress [ 44 ]. These findings suggested an association between shear stress and cell adhesion molecules (CAM) proteins, including CD56. Flow cytometry analysis showed a noticeable rightward shift of peaks in all groups under static conditions, indicating a statistically significant difference compared to the control under static conditions (Fig. 7 B). The extent of the peak shift was most pronounced in the group co-cultured with stressed feeder cells under dynamic conditions, showing a significant difference from the group co-cultured with non-stressed feeder cells. The flow cytometry results were consistent with those of RT-qPCR. In each category, the groups under static conditions showed peaks that shifted to the left compared with the groups under dynamic conditions. This is likely attributable to the influence of culture conditions, as described earlier, indicating that CD56 expression is affected not only by 4-1BB/4-1BBL binding but also by the culture environment. However, the peak of NK cells co-cultured with stressed feeder cells under static conditions shifted to a level similar to that observed under dynamic conditions, suggesting that 4-1BB/4-1BBL binding alone was sufficient to upregulate CD56 expression. These outcomes are consistent with the previously discussed RT-qPCR results, underscoring the similarity in CD56 expression between NK cells and stressed feeder cells under static conditions compared to those under dynamic conditions on Day 4. Furthermore, NK cells co-cultured with stressed feeder cells under dynamic conditions exhibited the most substantial upregulation of CD56 expression at both gene and protein levels. This observation is promising as it indicates that even under dynamic culture conditions, such as in bioreactors, feeder cells have the potential to induce enhanced NK cell proliferation. NK cells with elevated CD56 expression are recognized for their ability to stimulate the release of cytokines, including IFN-γ, TNF-α, and IL-10 [ 2 , 16 , 17 ]. Relevant experiments were conducted using RT-qPCR with gene markers associated with cytokines ( IFN-γ , TNF-α , and IL-10 ), as detailed in Supplementary Fig. 2. The experimental results demonstrated a more pronounced upregulation of expression under dynamic conditions than under static conditions across all categories. Bing Hu et al. reported that fluid shear stress (FSS) induces NK cell activation via NKG2D-mediated mechanosensing, leading to elevated levels of cytokine release [ 45 ]. The higher cytokine expression observed under dynamic conditions, irrespective of feeder cell presence, suggests that the upregulation of cytokine expression through NKG2D-mediated mechanosensing by shear stress had a more substantial impact on the RT-qPCR results than the upregulation of cytokine expression through CD56 upregulation. CONCLUSION In this study, extracellular stresses, such as hypoxia and cytochalasin D, were applied to Colo-205 cells to engineer feeder cells expressing the required ligand at an elevated level. After treatment with the stress factors, several analyses were performed to assess their efficacy. The results indicated that the density of feeder cells decreased by approximately 35.58%, expression of the proliferation-related gene ( MCM2 ) decreased by approximately 0.1-fold, expression of the stress-related gene ( MICA ) increased by approximately 4.04-fold, exosome release decreased by 11.67%, and expression of TNFSF9 (4-1BBL) increased by approximately 4.15-fold. After modification of the feeder cells, co-culture with NK cells was performed, and various analyses were carried out. During this phase, the culture conditions were divided into two types, namely, static and dynamic, to assess their applicability to environments subjected to shear stress. The results showed that NK cells co-cultured with stressed feeder cells under dynamic conditions exhibited the most substantial cell expansion, with approximately a 2.34-fold increase compared to the control under static conditions. However, as previously mentioned, the group cultured under dynamic conditions exhibited a substantial impact of the dynamic culture, suggesting a lack of significant differentiation of the feeder cells. This observation was supported by the results, which indicated only a marginal difference in cell numbers between the groups cultured under dynamic conditions on day 4. Additionally, the expression of proliferation-related genes was downregulated compared to that recorded on day 2. However, NK cells co-cultured with stressed feeder cells under static conditions exhibited significant cell expansion, with a cell number approximately 1.41-fold higher than that of the control under static conditions. This suggests that feeder cells significantly influenced cell expansion. Moreover, when assessing the expression of CD56 at both the gene and protein levels, it was confirmed that the peak position on the histogram did not show significant differences between NK cells co-cultured with stressed feeder cells under static and dynamic conditions. Thus, 4-1BB/4-1BBL binding via feeder cells significantly contributes to promoting NK cell proliferation. These findings present an appealing strategy for NK cell mass production, as it can be easily implemented without resorting to risky methods such as gene editing. Declarations ACKNOWLEDGMENTS This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (RS-2023-00207801, NRF-2021R1I1A3055700) and an Inha University Research Grant, Korea. Conflict of interest no conflict of interests Ethical Approval Not applicable Dada Availability Not applicable (this manuscript does not report data generation or analysis) References Spits H, Bernink JH, Lanier L (2016) NK cells and type 1 innate lymphoid cells: partners in host defense. Nat Immunol 17, 758-764. Cooper MA, Fehniger TA, Turner SC, Chen KS, Ghaheri BA, Ghayur T, Carson WE, Caligiuri MA (2001) Human natural killer cells: a unique innate immunoregulatory role for the CD56bright subset. Blood 97, 3146-3151. Kumar V, McNerney ME (2005) A new self: MHC-class-I-independent Natural-killer-cell self-tolerance. Nature Reviews Immunology 5, 363-374. Vivier E, Ugolini S, Blaise D, Chabannon C, Brossay L (2012) Targeting natural killer cells and natural killer T cells in cancer. Nat Rev Immunol 12, 239-252. 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J Extracell Vesicles 9, 1703244. Qiu L, Ning H, Zhu Y, Yang Q, Liu L, Luo L, Gao Y, Xing Y (2022) Feedback regulation of antioxidant transcription factor NFE2L1 and immunostimulatory factor 41BBL mediates the crosstalk between oxidative stress and tumor immunity. Molecular Immunology 141, 265-272. Du H, Bartleson JM, Butenko S, Alonso V, Liu WF, Winer DA, Butte MJ (2023) Tuning immunity through tissue mechanotransduction. Nat Rev Immunol 23, 174-188. Meng KP, Majedi FS, Thauland TJ, Butte MJ (2020) Mechanosensing through YAP controls T cell activation and metabolism. Journal of Experimental Medicine 217. Cooper MA, Fehniger TA, Caligiuri MA (2001) The biology of human natural killer-cell subsets. Trends Immunol 22, 633-640. Wang F, Wang Z, Pu J, Xie X, Gao X, Gu Y, Chen S, Zhang J (2019) Oscillating flow promotes inflammation through the TLR2–TAK1–IKK2 signalling pathway in human umbilical vein endothelial cell (HUVECs). Life Sciences 224, 212-221. Schwankhaus N, Gathmann C, Wicklein D, Riecken K, Schumacher U, Valentiner U (2014) Cell adhesion molecules in metastatic neuroblastoma models. Clin Exp Metastasis 31, 483-496. Hu B, Xin Y, Hu G, Li K, Tan Y (2023) Fluid shear stress enhances natural killer cell's cytotoxicity toward circulating tumor cells through NKG2D-mediated mechanosensing. APL Bioeng 7, 036108. Additional Declarations No competing interests reported. Supplementary Files SUPPLEMENTARYINFORMATION.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3918793","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":271918451,"identity":"aa1f52cb-d933-48e3-9169-53936979d571","order_by":0,"name":"Donghyun Lee","email":"","orcid":"","institution":"Inha University","correspondingAuthor":false,"prefix":"","firstName":"Donghyun","middleName":"","lastName":"Lee","suffix":""},{"id":271918452,"identity":"81aaea49-4557-4839-9936-fb2045e19aa8","order_by":1,"name":"Myeongkwan Song","email":"","orcid":"","institution":"Inha University","correspondingAuthor":false,"prefix":"","firstName":"Myeongkwan","middleName":"","lastName":"Song","suffix":""},{"id":271918453,"identity":"560ba1ca-9656-4fa0-b933-ffadd92799af","order_by":2,"name":"Soonjo Kwon","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIie2RIQvCQBTH3zGYRbA+MfgVbgwOg7Kv4hBmWTAuDoSzKFb9FkaDQRnMcpgXDFtZMmi0+aYIpnNR8H7l4Hi/u/97D8Bg+FUQgNPB8iHsnxcsrqlYvL4CL8UmtYbSnU3TvBeBy4+LNCq25y40kpytd5rXVTrmqEBwdQoyX5VO3Aw425QaBUOBbQl9kYUi82VCmUIaxF4TbPWhTEjx4tZFr0D2UkSlACl+jPTLRqNQLwGiQtdTykVSRhJLfljpgtHEOhj1nfVs7tzuMhksW6OimOuCERZWy3xjw3s7Gtj1W4XBYDD8Nw90alJT1FqOAAAAAABJRU5ErkJggg==","orcid":"","institution":"Inha University","correspondingAuthor":true,"prefix":"","firstName":"Soonjo","middleName":"","lastName":"Kwon","suffix":""}],"badges":[],"createdAt":"2024-02-01 21:29:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3918793/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3918793/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50998691,"identity":"ec2a7f25-01a8-4993-8ff8-08fbb3a7f87c","added_by":"auto","created_at":"2024-02-12 12:48:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":911693,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAn overview of the NK cell mass production strategy using stress-induced feeder cells. \u003c/strong\u003eBy subjecting feeder cells to hypoxia and cytochalasin D treatment, we observed an upregulation in the expression of 4-1BBL on the feeder cell surface, along with increased expression of stress-related ligands such as MICA/B and ULBP1. Subsequent co-culture with NK cells resulted in enhanced NK cell proliferation, facilitated by the interaction between NK cells and feeder cells.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3918793/v1/7d5dc724ae7895345746cdb4.png"},{"id":50998692,"identity":"eafeaec8-951a-4d65-91ec-e3ee09e7d009","added_by":"auto","created_at":"2024-02-12 12:48:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1269941,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of hypoxia and cytochalasin D treatment on the proliferation of feeder cells. \u003c/strong\u003eWhen cultured under normal conditions for three days following 24 h treatment with hypoxia and cytochalasin D, (A) cell density measurements revealed a decrease in the treated group compared to the control, with the gray dotted line indicating the seeding density of the feeder cells. (B) RT-qPCR analysis was conducted to assess the impact of stress factors on feeder cell proliferation, confirming a downregulation in the expression of proliferation-related marker genes (\u003cem\u003eMKI67\u003c/em\u003e, \u003cem\u003ePCNA\u003c/em\u003e, and \u003cem\u003eMCM2\u003c/em\u003e) in the treated group.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3918793/v1/a45698834217b095cf8052e7.png"},{"id":50998693,"identity":"df0a23c4-9bc7-47cd-82f2-1499f58fa658","added_by":"auto","created_at":"2024-02-12 12:48:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":149604,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlterations in the expression of stress-related ligand genes in feeder cells were examined after exposure to hypoxia and cytochalasin D treatment. \u003c/strong\u003eTo ascertain changes in the expression of stress-related ligands in feeder cells, RT-qPCR was conducted using relevant marker genes (\u003cem\u003eMICA\u003c/em\u003e, \u003cem\u003eMICB\u003c/em\u003e, and \u003cem\u003eULBP1\u003c/em\u003e). The results confirmed a significant upregulation of these markers in the group exposed to stress factors.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3918793/v1/358372dee2200b7d999762e9.png"},{"id":50998695,"identity":"27925295-1279-4245-a406-a18eada4a3fe","added_by":"auto","created_at":"2024-02-12 12:48:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":120381,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlterations in exosome release from feeder cells in response to hypoxia and cytochalasin D treatment. \u003c/strong\u003eTo investigate the impact of inhibiting exosome release from feeder cells following cytochalasin D treatment, we conducted an analysis using Nanoparticle Tracking Analysis (NTA). The experimental groups were categorized into hypoxia treatment alone, cytochalasin D treatment alone, and combined treatment with hypoxia and cytochalasin D. The results demonstrated that exosome secretion was inhibited in the group treated with cytochalasin D.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3918793/v1/bf4d4f3028b37a9aab233003.png"},{"id":50998698,"identity":"05fd0ad8-dbea-4644-a004-144bd659f003","added_by":"auto","created_at":"2024-02-12 12:48:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":117646,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlterations in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTNFSF9\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene (4-1BBL) expression within feeder cells in response to hypoxia and cytochalasin D treatment. \u003c/strong\u003eTo validate the enhancement in 4-1BBL expression due to hypoxia and cytochalasin D treatment, RT-qPCR was employed using \u003cem\u003eTNFSF9\u003c/em\u003e as the marker gene. The analysis confirmed a consistent upregulation of expression across all treated groups.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3918793/v1/37805aa0d8ca27ea362478a5.png"},{"id":50998696,"identity":"a7a00942-199d-4628-ac3a-e34feee55661","added_by":"auto","created_at":"2024-02-12 12:48:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2172757,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnhanced NK cell proliferation upon co-culturing with feeder cells. \u003c/strong\u003eAfter co-culturing feeder cells and NK cells, the enhancement of NK cell proliferation was validated through (A) an increase in relative cell number. For RT-qPCR analysis, we selected (B) \u003cem\u003eMKI67\u003c/em\u003e, (C) \u003cem\u003ePCNA\u003c/em\u003e, and (D) \u003cem\u003eMCM2\u003c/em\u003e as marker genes. The results confirmed the upregulation of the expression of proliferation-related genes.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3918793/v1/687151fa4c7e4cceb133d7fe.png"},{"id":50998697,"identity":"dd427f0e-530a-41ef-806d-9b46a1428ba8","added_by":"auto","created_at":"2024-02-12 12:48:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1946752,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUpregulation of CD56 expression on NK cells upon co-culturing with feeder cells. \u003c/strong\u003eThe elevation in CD56 expression in NK cells following the co-culture of feeder cells with NK cells was validated through (A) RT-qPCR, utilizing the \u003cem\u003eCD56\u003c/em\u003e gene as the marker gene to confirm the upregulation of expression at the gene level, and (B) flow cytometry using anti-CD56 antibody (APC conjugate) to confirm the upregulation of expression at the protein level.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3918793/v1/8c706f8a9d0fbd425a161fff.png"},{"id":51809442,"identity":"0480f201-b8de-441b-96ce-8308c5246cc1","added_by":"auto","created_at":"2024-02-29 11:55:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1594862,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3918793/v1/7b95d3d2-72a1-4c64-9232-59384e182278.pdf"},{"id":50999239,"identity":"d581c0db-6e06-453a-af0f-10380bfa3980","added_by":"auto","created_at":"2024-02-12 12:56:45","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":640662,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTARYINFORMATION.docx","url":"https://assets-eu.researchsquare.com/files/rs-3918793/v1/ae3c12d6bc720bbfbf3e08f5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhanced NK cell proliferation by stress-induced feeder cells","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eNatural Killer (NK) cells are prototypic members of the innate lymphoid cell (ILC) family, and in humans, NK cells are phenotypically characterized by being CD3\u003csup\u003e\u0026minus;\u003c/sup\u003e and CD56\u003csup\u003e+\u003c/sup\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. They are predominantly found in the peripheral blood, constituting 5\u0026ndash;15% of peripheral blood lymphocytes. According to the missing-self hypothesis, NK cells identify target cells by detecting the presence or absence of major histocompatibility complex (MHC) class I molecules [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In contrast, T cells distinguish between self and non-self based on the structure of MHC class I, initiating an attack on the target cell if it is identified as non-self. Essentially, this limitation limits the application of T-cell therapy to autologous applications. NK cells, unlike T cells, are not restricted by MHC class I molecules during target recognition, making them suitable for allogeneic cell therapy [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These advantages render NK cells compelling candidates for off-the-shelf cellular therapeutics. Consequently, recent efforts have focused on substituting NK cells with T cells as a source of cellular therapeutics. However, a significant drawback associated with NK cells is their limited half-life compared to other immune cells, which poses a substantial challenge in the proliferation in an in vitro setting [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. To address the limitations of NK cells, various approaches are underway, including gene editing [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and culturing in bioreactors [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Among these methods, co-culture with feeder cells is the most effective strategy for generating NK cells in vitro. This approach has been used to enhance NK cell proliferation.\u003c/p\u003e \u003cp\u003eThe proliferation NK cell relies on cell interactions where 4-1BB on NK cells binds to 4-1BBL on feeder cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The binding of 4-1BB/4-1BBL represents a cell-cell interaction that triggers the upregulation of NF-κB, ATF2, and c-Jun expression. This initiates a cascade of events that promotes NK cell proliferation, as evidenced in previous studies [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Most studies have achieved this via gene editing of feeder cells, a method associated with the drawback of potential induction of mutations during the process. Therefore, safer alternatives should be explored.\u003c/p\u003e \u003cp\u003eConsistent with numerous previous studies, the interaction between 4-1BB and 4-1BBL resulted in the upregulation of CD56 (neural cell adhesion molecule, NCAM-1) expression in NK cells. Notably, CD56\u003csup\u003ebright\u003c/sup\u003e NK cells, characterized by increased CD56 expression, exhibit more vigorous proliferation, as highlighted in previous studies [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. While the mechanism underlying CD56\u0026rsquo;s promotion of NK cell proliferation remains unclear, some researchers suggest that increased cytokine release as a potential contributing factor, corresponding with the upregulated expression of CD56 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, Colo-205 cells were selected as the preferred feeder cells among various candidates. Colo-205 tumor cells are known for their remarkably high levels of 4-1BBL expression [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Modifications were implemented to inhibit feeder cell proliferation without resorting to gene editing, involving pretreatment with cytochalasin D under hypoxic conditions. Furthermore, subjecting Colo-205 cells to extracellular stress during pretreatment resulted in the upregulation of 4-1BBL expression. Subsequently, we measured cell expansion to validate the enhanced proliferation of NK cells during coculture. Furthermore, we examined the phenotypic changes in NK cells. At this point, we divided the culture process into static and dynamic conditions to evaluate the feasibility of employing feeder cells in dynamic environments, such as bioreactors. Our findings revealed that pre-treated and modified Colo-205 cells, serving as feeder cells, significantly enhanced NK proliferation under both static and dynamic conditions.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eColo-205 human colon cancer cells were purchased from the Korea Cell Line Bank (KCLB Korea), and KHYG-1 human natural killer cells were purchased from AcceGen Biotech (ABC-TC0506, USA). Colo-205 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin at 37\u0026deg;C in a humidified CO\u003csub\u003e2\u003c/sub\u003e incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. KHYG-1 cells were cultured under identical conditions as Colo-205, supplemented with an additional 100 U/mL of recombinant human interleukin-2 (Peprotech, 200-02). Both cell types were seeded in a 90 \u0026times; 20 mm cell culture dish (SPL, 20101). The medium was changed every 3\u0026ndash;4 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eFeeder cell modification\u003c/h2\u003e \u003cp\u003eTo induce hypoxic conditions in Colo-205 cells for modification, sodium sulfite and cobalt chloride were utilized, while cytochalasin D was employed to enhance the efficiency of feeder cell utilization. Colo-205 cells were seeded at a density of 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e in a 60 \u0026times; 15 mm cell culture dish (SPL, 20060). Subsequently, the culture medium was replaced with 3 mL of fresh media supplemented with 4 mM sodium sulfite (Sigma), 100 \u0026micro;M cobalt chloride (Sigma), and 3 \u0026micro;M cytochalasin D (Cell Signaling Technology). Treated Colo-205 cells were then cultured for 24 h at 37\u0026deg;C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eExosome isolation methods\u003c/h2\u003e \u003cp\u003eTo analyze the inhibitory effect of cytochalasin D on exosomes, exosomes were isolated from cytochalasin-D-treated Colo-205 cells using a centrifugation-based method. Initially, the culture media of Colo-205 cells were collected and subjected to centrifugation at 2000 \u0026times; \u003cem\u003eg\u003c/em\u003e and 4\u0026deg;C twice. The supernatant obtained from this process was further subjected to ultracentrifugation at 100,000 \u0026times; \u003cem\u003eg\u003c/em\u003e and 4\u0026deg;C, following which the pellet containing exosomes was collected. Next, nanoparticle tracking analysis (NTA) was conducted using the ZetaView\u0026reg; PMX-220 (Particle Metrix GmbH, Germany) to quantify the concentration of exosomes released by Colo-205 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCo-culture of NK cells and feeder cells\u003c/h2\u003e \u003cp\u003eA T-75 flask (SPL 70075) was utilized for the co-culture of NK cells and feeder cells. Under dynamic conditions, the co-culture was carried out on a digital rocker RK-2D (DAIHAN, Korea) to subject the NK cells to shear stress. The RPM speed of the digital rocker was set to 10 RPM. The seeding cell number ratio between NK cells and feeder cells was set at 100:1. The culture period extended for four days. Additionally, on the second day of co-culture, the medium was changed to prevent NK cell proliferation caused by media depletion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eGene expression profiling by RT-qPCR\u003c/h2\u003e \u003cp\u003eRT-qPCR was performed to determine gene expression. The proliferation markers Ki-67 (\u003cem\u003eMKI67\u003c/em\u003e), proliferating cell nuclear antigen (\u003cem\u003ePCNA\u003c/em\u003e), and minichromosome maintenance complex component 2 (\u003cem\u003eMCM2\u003c/em\u003e), were chosen as gene indicators for cell proliferation. Stress-related genes in the feeder cells included those encoding for MHC class I polypeptide-related sequence A (\u003cem\u003eMICA\u003c/em\u003e), MHC class I polypeptide-related sequence B (\u003cem\u003eMICB\u003c/em\u003e), and UL16 binding protein 1 (\u003cem\u003eULBP1\u003c/em\u003e). Genes encoding for tumor necrosis factor superfamily member 9 (\u003cem\u003eTNFSF9\u003c/em\u003e) and tumor necrosis factor receptor superfamily member 9 (\u003cem\u003eTNFRSF9\u003c/em\u003e) were selected as genes related to 4-1BBL and 4-1BB in feeder and NK cells. The CD56 gene was chosen to indicate the expression of its corresponding protein in NK cells. \u003cem\u003eIFN-γ\u003c/em\u003e, \u003cem\u003eTNF-α\u003c/em\u003e, and \u003cem\u003eIL-10\u003c/em\u003e were chosen as genes representing cytokines in NK cells, with glyceraldehyde 3-phosphate dehydrogenase (\u003cem\u003eGAPDH\u003c/em\u003e) serving as the housekeeping gene (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To isolate total cellular mRNA from K562 cells, the cells were treated with the TRIzol reagent in cell plates for 5 min. Trizol reagent and cellular mRNA were then transferred to microcentrifuge tubes, and 200 \u0026micro;L of chloroform was added, followed by centrifugation at 12,000 \u0026times; \u003cem\u003eg\u003c/em\u003e and 4 ℃ for 10 min. The supernatant was then transferred to a new microcentrifuge tube. To the tube, an equal volume of isopropanol was added, followed by additional centrifugation at 12,000 \u0026times; \u003cem\u003eg\u003c/em\u003e and 4 ℃ for 5 min. The supernatant was then discarded, and 75% ethanol was added to the tube before further centrifugation at 12,000 \u0026times; \u003cem\u003eg\u003c/em\u003e and 4 ℃ for 5 min. The supernatant was removed and air-dried. To dissolve the mRNA pellet, RNase-free water was added, and the mixture was incubated at 60 ℃ for 10 min. The mRNA concentration was quantified using a Nanodrop DS-11 (DeNovix, Wilmington, DE, USA). To synthesize cDNA from mRNA (1 \u0026micro;g), we used a cDNA synthesis kit (Takara) according to the manufacturer\u0026rsquo;s protocol. The synthesized cDNA was mixed with TB Green\u0026reg; Premix Ex Taq\u0026trade; II (Takara), the appropriate target gene primer, and RNase-free water, after which RT-qPCR was performed. The primer sequences are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Fold changes in target gene expression levels were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimer sequences\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTarget gene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSize\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGAPDH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF : 5\u0026rsquo;-GAAATCCCATCACCATCTTCCAGG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eR : 5\u0026rsquo;-GAGCCCCAGCCTTCTCCATG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMKI67\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF : 5\u0026rsquo;-CGTCCCAGTGGAAGAGTTGT-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eR : 5\u0026rsquo;-CGACCCCGCTCCTTTTGATA-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e143\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePCNA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF : 5\u0026rsquo;-CGGTTACTGAGGGCGAGAAG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eR : 5\u0026rsquo;-GCTGAGACTTGCGTAAGGGA-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMCM2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF : 5\u0026rsquo;-ATCGTGGTACTGCTATGGCG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eR : 5\u0026rsquo;-TGGAGGTGAGGGCATCAGTA-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e135\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMICA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF : 5\u0026rsquo;-CTAAAGTCTGAGAGAGGGAAGTCG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eR : 5\u0026rsquo;-CGAAGACTGTGGGGCTCTTATAC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMICB\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF : 5\u0026rsquo;-TAAAGTCTGCGAGGAGGAAGTCG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eR : 5\u0026rsquo;-GACTGCACAGATCCATCCTGG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eULBP1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF : 5\u0026rsquo;-GACAGCACTTCATCCTGGAGC-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eR : 5\u0026rsquo;-CAGAGAGGGTGGTTTTGTTGGA-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e166\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTNFSF9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF : 5\u0026rsquo;-CCAGGCTAGGGGGCTATAGAA-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eR : 5\u0026rsquo;-GAGGCTGGGGATGAGTCTTT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTNFRSF9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF : 5\u0026rsquo;-CGTTGCTCTTCCTGCTGTTC-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eR : 5\u0026rsquo;-CTTCTTCTGGAAATCGGCAGC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e153\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCD56\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF : 5\u0026rsquo;-CACTTATCGCTGTGAGGGCA-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eR : 5\u0026rsquo;-TTTGTCCAGCTCATGGTGGG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e192\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIFN-γ\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF : 5\u0026rsquo;-GAGTGTGGAGACCATCAAGGAAG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eR : 5\u0026rsquo;-TGCTTTGCGTTGGACATTCAAGTC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e124\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTNF-α\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF : 5\u0026rsquo;-CGAGTGACAAGCCTGTAGCC-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eR : 5\u0026rsquo;-GGACCTGGGAGTAGATGAGGT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e165\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIL-10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF : 5\u0026rsquo;-GCAAAACCAAACCACAAGACAG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eR : 5\u0026rsquo;-TCAGGAGGACCAGGCAACAG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eProtein expression profile study by flow cytometry\u003c/h2\u003e \u003cp\u003eTo assess CD56 expression, flow cytometry was conducted following the manufacturer\u0026rsquo;s protocol of the APC-conjugated anti-human CD56 antibody (Cell Signaling Technology). Post co-culturing feeder cells and NK cells, washing was carried out using 0.5% BSA (Sigma) DPBS buffer at 182 \u0026times; \u003cem\u003eg\u003c/em\u003e and 4 ℃ for 3 min. Following this, the APC-conjugated anti-human CD56 antibody was diluted at a ratio of 20:1 in 0.5% BSA DPBS and added to the NK cells, followed by incubation on ice for 60 min. The cells were subsequently washed twice with 0.5% BSA DPBS before flow cytometry analysis. Stained cells were analyzed using a BD\u0026reg; LSR II Flow Cytometer (BD Biosciences, USA) employing the FACSDiva v9.0 software (BD Biosciences, USA). Data analysis was performed using the FlowJo\u0026trade; v10.8 software (BD Biosciences, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eCollected data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (standard error of the mean (SEM). Differences between the control and experimental groups were assessed using the Student\u0026rsquo;s t-test. Differences were considered statistically significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. GraphPad Prism v10.1.2 software (GraphPad, USA) was used for statistical analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eInhibition of feeder cell proliferation by stress factors\u003c/h2\u003e \u003cp\u003eTo improve the effectiveness of Colo-205 cells as feeder cells, they underwent a 24 h treatment with cytochalasin D under hypoxic conditions, followed by three days of culturing under normal conditions. Subsequently, viable cell density was assessed using a trypan blue assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). All groups, including the hypoxia-alone, cytochalasin D-alone, and combined hypoxia and cytochalasin D groups, exhibited lower cell densities compared to the control. Each group demonstrated viable cell densities of approximately 89.46%, 48.52%, and 35.58%, respectively, compared with the control. The results were considered statistically significant.\u003c/p\u003e \u003cp\u003eAdditionally, we aimed to evaluate the impact of these stress factors on Colo-205 cell proliferation by examining marker genes associated with proliferation through RT-qPCR. The selected markers included \u003cem\u003eMKI67\u003c/em\u003e, which indicates ongoing cell cycle progression [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], \u003cem\u003ePCNA\u003c/em\u003e, involved in forming a protein complex crucial for DNA replication during the DNA synthesis phase [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and \u003cem\u003eMCM2\u003c/em\u003e, which participates in DNA replication initiation by interacting with the origin recognition complex (ORC) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], the initiator of DNA replication. RT-qPCR analysis was conducted for these markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The experiment yielded conclusive results, indicating a statistically significant downregulation in the expression of all three genes compared to that in the control across all groups. In the combined hypoxia and cytochalasin D group, \u003cem\u003eMKI67\u003c/em\u003e expression decreased by 0.33-fold, \u003cem\u003ePCNA\u003c/em\u003e by 0.52-fold, and \u003cem\u003eMCM2\u003c/em\u003e by 0.10-fold compared to the control group. These RT-qPCR results regarding proliferation-related markers corroborated the findings from the trypan blue assay described earlier.\u003c/p\u003e \u003cp\u003eThe results of the two experiments showed consistent tendencies. Specifically, it was evident that cell density was significantly decreased and gene expression was downregulated, particularly in the groups treated with cytochalasin D. The variations in results between groups treated with and without cytochalasin D can be attributed to the distinct mechanisms through which each stress factor induces cell cycle arrest. HIF-1α accumulates within cells under hypoxic conditions, is known to regulate the p27 expression, thereby preventing cells in the G1 phase from entering the S phase [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, under normoxic conditions, the accumulated HIF-1α is decomposed, allowing the cell cycle to proceed normally again [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, the decrease in cell density and expression of proliferation-related markers in the hypoxia-only treatment group was less pronounced than in the other groups. Cytochalasin D impedes cell cycle progression by inhibiting actin polymerization within cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This action primarily affects the M phase, necessitating actin polymerization and inhibiting the cell membrane formation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Unlike the accumulation of HIF-1α under hypoxic conditions, the effects of cytochalasin D persisted even under normal conditions. Consequently, cell density and the expression of proliferation-related genes remained consistently low, particularly in the groups treated with cytochalasin D.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eModulation of NKG2DL expression in feeder cells induced by stress factor\u003c/h2\u003e \u003cp\u003eThe expression of NKG2DL, such as MICA/B and ULBP1-6, is upregulated in abnormal cells, such as cancer cells [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Additionally, the expression is also upregulated when extracellular stress is applied to these abnormal cells [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. When NKG2DL binds to the NKG2D receptor on the surface of NK cells, NKG2D is activated. Activated NKG2D plays a crucial role in promoting cell proliferation through various signaling pathways [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. To confirm alterations in the expression of stress-related ligands in feeder cells under stress conditions such as hypoxia, cytochalasin D, \u003cem\u003eMICA\u003c/em\u003e, \u003cem\u003eMICB\u003c/em\u003e, and \u003cem\u003eULBP1\u003c/em\u003e as marker genes for stress-related marker genes, and RT-qPCR was conducted (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The experiment confirmed that the expression was upregulated in all groups compared to that in the control group. Especially in the group subjected to both hypoxia and cytochalasin D group, the \u003cem\u003eMICA\u003c/em\u003e exhibited a 4.04-fold increase, \u003cem\u003eMICB\u003c/em\u003e showed a 2.94-fold increase, and \u003cem\u003eULBP1\u003c/em\u003e demonstrated a 1.61-fold increase, signifying a substantial elevation due to the other stress factors employed in this study. Additionally, the experimental results were statistically significant for all outcomes except \u003cem\u003eULBP1\u003c/em\u003e in the hypoxia-alone group. In addition to NK cell activation through 4-1BB/4-1BBL binding mentioned in the introduction we anticipate the promotion of NK cell proliferation through the signaling pathway mediated by NKG2D/NKG2DL binding between NK cells and feeder cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eInhibition of exosome release in feeder cells mediated by stress factors\u003c/h2\u003e \u003cp\u003eUnder hypoxic conditions, the release of exosomes by tumor cells is higher than that under normal conditions [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Furthermore, tumor exosomes released under hypoxic conditions actively inhibit NK cell function via NKG2DL on the exosome surface [\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. \u003cem\u003eMilad Moloudizargari\u003c/em\u003e et al. reported that NKG2D, which normally binds to MICA/B on the tumor cell surface, binds to MICA on the surface of tumor exosomes. This enables tumor cells to evade NK-mediated antitumor immunity through exosomal MICA/B shedding, a phenomenon termed immune escape [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. \u003cem\u003eGuy Berchem\u003c/em\u003e et al. reported that tumor exosomes inhibit the function of NKG2D via the expression of TGF-β [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Hence, to prevent the inhibition of NK cell function by tumor exosomes, it is imperative to suppress exosome release from tumor cells for use as feeder cells. Cytochalasin D is known to inhibit actin polymerization and is known for its ability to block exosome release [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Thus, to maintain optimal NK cell function, we aimed to create feeder cells with suppressed exosome release after cytochalasin D treatment. Therefore, to determine the effect of cytochalasin D treatment on exosome release from feeder cells, we isolated exosomes from feeder cells by ultracentrifugation and analyzed them by NTA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e). When the quantified data were analyzed, the exosome concentrations for each group were as follows: Control, 1.2 \u0026times; 10\u003csup\u003e10\u003c/sup\u003e particles/mL; hypoxia-alone group, 2.0 \u0026times; 10\u003csup\u003e10\u003c/sup\u003e particles/mL; cytochalasin D-alone group, 1.9 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e particles/mL; and the combined hypoxia and cytochalasin D group, 1.4 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e particles/mL. Compared with the control, the exosome release rates were approximately 166.67%, 15.83%, and 11.67%, respectively. Specifically, the group treated with hypoxia alone exhibited a 1.67-fold higher exosome concentration than the control. This result aligns with the concept that hypoxia induces tumor cells to release more exosomes, as explained earlier. Furthermore, the two groups treated with cytochalasin D exhibited comparable exosome concentrations. This similarity arises from the fact that actin polymerization was already inhibited by cytochalasin D in these groups, leading to the inhibition of exosome release regardless of the application of hypoxia.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEnhanced 4-1BBL expression in feeder cells induced by stress factors\u003c/h2\u003e \u003cp\u003eVarious tumor cells express 4-1BBL. According to \u003cem\u003eHelmut R. Salih\u003c/em\u003e et al., Colo-205 cells exhibit the highest levels of 4-1BBL expression [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. To evaluate the impact of stress factors on the expression of 4-1BBL in feeder cells, \u003cem\u003eTNFSF9\u003c/em\u003e (4-1BBL) was selected as a marker, and RT-qPCR was conducted (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003e). All groups exhibited a statistically significant upregulation of \u003cem\u003eTNFSF9\u003c/em\u003e expression compared to the control, with specific fold changes as follows: 1.70-fold in the hypoxia-only group, 3.52-fold in the cytochalasin D-only group, and 4.15-fold in the combined hypoxia and cytochalasin D group compared to the control. While the correlation between extracellular stress and the expression of 4-1BBL has not been established, \u003cem\u003eLu Qiu\u003c/em\u003e et al. reported that cells subjected to oxidative stress exhibit increased 4-1BBL gene transcription. This effect is mediated by the intracellular antioxidant transcription factor NFE2L1, which promotes 4-1BBL gene transcription via ARE in the promoter region [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFeeder cells, following this modification, were co-cultured with NK cells to induce 4-1BB/4-1BBL binding. Subsequently, RT-qPCR was performed using \u003cem\u003eTNFRSF9\u003c/em\u003e (4-1BB) as the selected marker (Supplementary Fig.\u0026nbsp;1). In summary, the co-culture group under dynamic conditions exhibited a more pronounced upregulation of \u003cem\u003eTNFRSF9\u003c/em\u003e (4-1BB) expression than the co-culture group under static conditions. Additionally, \u003cem\u003eTNFRSF9\u003c/em\u003e (4-1BB) expression increased in the control group under dynamic conditions even in the absence of 4-1BB/4-1BBL binding. These results support the notion that regulation of 4-1BB expression in NK cells is influenced not only by 4-1BB/4-1BBL binding but also by the prevailing culture conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEnhanced proliferation of NK cells following co-culture with feeder cells\u003c/h2\u003e \u003cp\u003eWe evaluated the extent to which NK cell proliferation enhancement by co-culturing the feeder cells previously subjected to modifications with NK cells. The experiments were categorized into three groups: NK cells cultured independently without feeder cells (control), NK cells co-cultured with unstressed feeder cells (non-stressed feeder cells), and NK cells co-cultured with stress-induced feeder cells (stressed feeder cells). To assess distinctions in the culture methods for each group, the cultures were segregated into static and dynamic conditions, with a total culture period of four days.\u003c/p\u003e \u003cp\u003eInitially, we performed a trypan blue assay on NK cells after co-culturing with feeder cells to assess the extent of cell number expansion facilitated by the feeder cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Under dynamic conditions, all groups exhibited over a 2-fold increase in cell expansion compared to the control under static conditions (approximately 2.17-fold, 2.18-fold, and 2.34-fold), with all results reaching statistical significance compared to the control. Conversely, NK cells co-cultured with non-stressed feeder cells under static conditions displayed a cell number comparable to that of control cells (approximately 0.96-fold). However, NK cells co-cultured with stressed feeder cells under static conditions exhibited statistically significant cell expansion compared with the control (approximately 1.41-fold). This results indicate a significant increase in cell number compared with NK cells co-cultured with non-stressed feeder cells under static conditions.\u003c/p\u003e \u003cp\u003eSubsequently, to assess the impact of co-culture with feeder cells on NK cell proliferation, we conducted RT-qPCR using the proliferation-related genes \u003cem\u003eMKI67\u003c/em\u003e, \u003cem\u003ePCNA\u003c/em\u003e, and \u003cem\u003eMCM2\u003c/em\u003e as markers [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. RT-qPCR was conducted using samples collected on days 2 and 4 of the incubation period (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-D).\u003c/p\u003e \u003cp\u003eOn Day 2, the expression of the \u003cem\u003eMKI67\u003c/em\u003e gene was significantly upregulated in all groups, except for NK cells with non-stressed feeder cells in static conditions, compared to their respective controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Furthermore, the expression on Day 4 was significantly upregulated compared to the respective controls in all groups, except for the two groups of NK cells with non-stressed feeder cells under static and dynamic conditions. The disparity in \u003cem\u003eMKI67\u003c/em\u003e expression in NK cells resulting from feeder cell exposure to stress factors was notably and statistically significantly upregulated in NK cells with stressed feeder cells under static conditions on both days 2 and 4.\u003c/p\u003e \u003cp\u003eOn day 2, the expression of the \u003cem\u003ePCNA\u003c/em\u003e gene showed significant upregulation in all groups, except for NK cells co-cultured with stressed and non-stressed feeder cells under static conditions, compared to their respective controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Furthermore, the expression on day 4 was significantly upregulated compared to the respective controls in all groups. The disparity in \u003cem\u003ePCNA\u003c/em\u003e expression in NK cells resulting from feeder cell exposure to stress factors was notably and statistically significantly increased in NK cells with stressed feeder cells under static conditions on both days 2 and 4. Moreover, proliferation-related gene expression was downregulated in most cases in NK cells with and without feeder cells under dynamic conditions. A notable exception was found in the expression of PCNA in NK cells with non-stressed feeder cells under dynamic conditions, exhibiting a slight upregulation on day 4. Given that PCNA is involved in DNA damage repair, this upregulation suggests a potential response to repair the damage incurred by NK cells owing to their interaction with non-stressed feeder cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAt the end of the Day 2 incubation period, the expression of the \u003cem\u003eMCM2\u003c/em\u003e gene was significantly upregulated in all groups, except for NK cells with non-stressed feeder cells under static conditions, compared to their respective controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Furthermore, the expression on day 4 was significantly upregulated compared to the respective controls in all groups. The significant upregulation of \u003cem\u003eMCM2\u003c/em\u003e expression in NK cells co-cultured with stressed feeder cells under static conditions on both days 2 and 4.\u003c/p\u003e \u003cp\u003eWhen comparing the results, no statistically significant differences were observed between NK cells with and without feeder cells in dynamic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The proliferative enhancement effect induced by dynamic conditions on day 4 was deemed more significant comparatively to the impact of feeder cell presence, explaining the observed attribution. \u003cem\u003eHuixun Du\u003c/em\u003e et al. reported two pathways for the enhancement of proliferation by externally applied shear stress: 1) Inhibition of the Hippo pathway, and 2) activation of transient receptor potential vanilloid type 4 (TRPV4) and piezo-type mechanosensitive ion channel component 1 (PIEZO1) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Under shear stress conditions, inhibition of the Hippo pathway occurs, facilitating the translocation of phosphorylated Yes-associated protein (YAP) and PDZ-binding motif (TAZ) into the nucleus, thereby promoting the proliferation of immune cells [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. \u003cem\u003eKevin P. Meng\u003c/em\u003e et al. reported that the activation of YAP inhibits T-cell activation and proliferation, supporting the enhancement mentioned earlier, of proliferation through the inhibition of the Hippo pathway [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Subsequently, findings, it was observed that the application of shear stress to cells activates the cation channels TRPV4 and PIEZO1. This activation leads to an augmented influx of Ca\u003csup\u003e2+\u003c/sup\u003e, and the heightened Ca\u003csup\u003e2+\u003c/sup\u003e influx, in turn, facilitates cell proliferation by regulating transcription factors [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eComparing the results on day 2, there was a more pronounced upregulation when cells were cultured under dynamic conditions compared to static conditions the expression of genes associated with proliferation exhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-D). These results further corroborate that the impact of dynamic conditions on proliferation was relatively more substantial than the influence of the presence of feeder cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCD56 expression of NK cells following co-culture with feeder cells\u003c/h2\u003e \u003cp\u003eUpon engaging in 4-1BB/4-1BBL binding, NK cells undergo activation, characterized by elevated levels of CD56 (NCAM-1) expression [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. To investigate changes in CD56 expression, we co-cultured NK cells with feeder cells previously subjected to modification, examining alterations at both the gene and protein levels in NK cells. The co-culture was performed according to a previously described procedure. RT-qPCR analyses were conducted using samples collected on days 2 and 4 of the culture period, with emphasis placed on the \u003cem\u003eCD56\u003c/em\u003e gene selected as the marker. Flow cytometry analysis was conducted on day 4, marking the completion of the culture. The anti-CD56 antibody (APC conjugate) was selected as a marker for this analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-B).\u003c/p\u003e \u003cp\u003eRT-qPCR analysis confirmed a statistically significant upregulation in expression levels in all groups, regardless of the culture method, exceeding that of the respective controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Moreover, a notable increase in CD56 expression in NK cells was observed upon the exposure of feeder cells to stress factors. Specifically, this upregulation was notable in NK cells with stressed feeder cells under static conditions on day 2 and in NK cells with stressed feeder cells under dynamic conditions on day 4. On Day 2, CD56 expression in the dynamic condition group exhibited more pronounced upregulation within each group. This observation suggests that the expression of CD56 is notably influenced by culture conditions, similar to the previously depicted impact on proliferation. \u003cem\u003eFeng Wang\u003c/em\u003e et al. reported an upregulation in the expression of cell adhesion molecule (CAM) proteins such as VCAM-1 and ICAM-1, similar to CD56, in human umbilical vein endothelial cells (HUVEC) subjected to shear stress [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Furthermore, \u003cem\u003eNina Schwankhaus\u003c/em\u003e et al. reported upregulation of ICAM-1 expression when SK-N-SH and LAN1 cells were exposed to shear stress [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. These findings suggested an association between shear stress and cell adhesion molecules (CAM) proteins, including CD56.\u003c/p\u003e \u003cp\u003eFlow cytometry analysis showed a noticeable rightward shift of peaks in all groups under static conditions, indicating a statistically significant difference compared to the control under static conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The extent of the peak shift was most pronounced in the group co-cultured with stressed feeder cells under dynamic conditions, showing a significant difference from the group co-cultured with non-stressed feeder cells. The flow cytometry results were consistent with those of RT-qPCR. In each category, the groups under static conditions showed peaks that shifted to the left compared with the groups under dynamic conditions. This is likely attributable to the influence of culture conditions, as described earlier, indicating that CD56 expression is affected not only by 4-1BB/4-1BBL binding but also by the culture environment. However, the peak of NK cells co-cultured with stressed feeder cells under static conditions shifted to a level similar to that observed under dynamic conditions, suggesting that 4-1BB/4-1BBL binding alone was sufficient to upregulate CD56 expression. These outcomes are consistent with the previously discussed RT-qPCR results, underscoring the similarity in CD56 expression between NK cells and stressed feeder cells under static conditions compared to those under dynamic conditions on Day 4.\u003c/p\u003e \u003cp\u003eFurthermore, NK cells co-cultured with stressed feeder cells under dynamic conditions exhibited the most substantial upregulation of CD56 expression at both gene and protein levels. This observation is promising as it indicates that even under dynamic culture conditions, such as in bioreactors, feeder cells have the potential to induce enhanced NK cell proliferation.\u003c/p\u003e \u003cp\u003eNK cells with elevated CD56 expression are recognized for their ability to stimulate the release of cytokines, including IFN-γ, TNF-α, and IL-10 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Relevant experiments were conducted using RT-qPCR with gene markers associated with cytokines (\u003cem\u003eIFN-γ\u003c/em\u003e, \u003cem\u003eTNF-α\u003c/em\u003e, and \u003cem\u003eIL-10\u003c/em\u003e), as detailed in Supplementary Fig.\u0026nbsp;2. The experimental results demonstrated a more pronounced upregulation of expression under dynamic conditions than under static conditions across all categories. \u003cem\u003eBing Hu\u003c/em\u003e et al. reported that fluid shear stress (FSS) induces NK cell activation via NKG2D-mediated mechanosensing, leading to elevated levels of cytokine release [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The higher cytokine expression observed under dynamic conditions, irrespective of feeder cell presence, suggests that the upregulation of cytokine expression through NKG2D-mediated mechanosensing by shear stress had a more substantial impact on the RT-qPCR results than the upregulation of cytokine expression through CD56 upregulation.\u003c/p\u003e \u003c/div\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn this study, extracellular stresses, such as hypoxia and cytochalasin D, were applied to Colo-205 cells to engineer feeder cells expressing the required ligand at an elevated level. After treatment with the stress factors, several analyses were performed to assess their efficacy. The results indicated that the density of feeder cells decreased by approximately 35.58%, expression of the proliferation-related gene (\u003cem\u003eMCM2\u003c/em\u003e) decreased by approximately 0.1-fold, expression of the stress-related gene (\u003cem\u003eMICA\u003c/em\u003e) increased by approximately 4.04-fold, exosome release decreased by 11.67%, and expression of \u003cem\u003eTNFSF9\u003c/em\u003e (4-1BBL) increased by approximately 4.15-fold. After modification of the feeder cells, co-culture with NK cells was performed, and various analyses were carried out. During this phase, the culture conditions were divided into two types, namely, static and dynamic, to assess their applicability to environments subjected to shear stress. The results showed that NK cells co-cultured with stressed feeder cells under dynamic conditions exhibited the most substantial cell expansion, with approximately a 2.34-fold increase compared to the control under static conditions. However, as previously mentioned, the group cultured under dynamic conditions exhibited a substantial impact of the dynamic culture, suggesting a lack of significant differentiation of the feeder cells. This observation was supported by the results, which indicated only a marginal difference in cell numbers between the groups cultured under dynamic conditions on day 4. Additionally, the expression of proliferation-related genes was downregulated compared to that recorded on day 2. However, NK cells co-cultured with stressed feeder cells under static conditions exhibited significant cell expansion, with a cell number approximately 1.41-fold higher than that of the control under static conditions. This suggests that feeder cells significantly influenced cell expansion. Moreover, when assessing the expression of CD56 at both the gene and protein levels, it was confirmed that the peak position on the histogram did not show significant differences between NK cells co-cultured with stressed feeder cells under static and dynamic conditions. Thus, 4-1BB/4-1BBL binding via feeder cells significantly contributes to promoting NK cell proliferation. These findings present an appealing strategy for NK cell mass production, as it can be easily implemented without resorting to risky methods such as gene editing.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (RS-2023-00207801, NRF-2021R1I1A3055700)\u0026nbsp;and an Inha University Research Grant, Korea.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eno conflict of interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eDada Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable (this manuscript does not report data generation or analysis)\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSpits H, Bernink JH, Lanier L (2016) NK cells and type 1 innate lymphoid cells: partners in host defense. \u003cem\u003eNat Immunol\u003c/em\u003e 17, 758-764.\u003c/li\u003e\n\u003cli\u003eCooper MA, Fehniger TA, Turner SC, Chen KS, Ghaheri BA, Ghayur T, Carson WE, Caligiuri MA (2001) Human natural killer cells: a unique innate immunoregulatory role for the CD56bright subset. \u003cem\u003eBlood\u003c/em\u003e 97, 3146-3151.\u003c/li\u003e\n\u003cli\u003eKumar V, McNerney ME (2005) A new self: MHC-class-I-independent Natural-killer-cell self-tolerance. \u003cem\u003eNature Reviews Immunology\u003c/em\u003e 5, 363-374.\u003c/li\u003e\n\u003cli\u003eVivier E, Ugolini S, Blaise D, Chabannon C, Brossay L (2012) Targeting natural killer cells and natural killer T cells in cancer. \u003cem\u003eNat Rev Immunol\u003c/em\u003e 12, 239-252.\u003c/li\u003e\n\u003cli\u003eZhang Y, Wallace DL, de Lara CM, Ghattas H, Asquith B, Worth A, Griffin GE, Taylor GP, Tough DF, Beverley PC, Macallan DC (2007) In vivo kinetics of human natural killer cells: the effects of ageing and acute and chronic viral infection. \u003cem\u003eImmunology\u003c/em\u003e 121, 258-265.\u003c/li\u003e\n\u003cli\u003eGurney M, Kundu S, Pandey S, O\u0026apos;Dwyer M (2022) Feeder Cells at the Interface of Natural Killer Cell Activation, Expansion and Gene Editing. \u003cem\u003eFront Immunol\u003c/em\u003e 13, 802906.\u003c/li\u003e\n\u003cli\u003eLi X, He C, Liu C, Ma J, Ma P, Cui H, Tao H, Gao B (2015) Expansion of NK cells from PBMCs using immobilized 4-1BBL and interleukin-21. \u003cem\u003eInt J Oncol\u003c/em\u003e 47, 335-342.\u003c/li\u003e\n\u003cli\u003eVidard L, Dureuil C, Baudhuin J, Vescovi L, Durand L, Sierra V, Parmantier E (2019) CD137 (4-1BB) Engagement Fine-Tunes Synergistic IL-15- and IL-21-Driven NK Cell Proliferation. \u003cem\u003eJ Immunol\u003c/em\u003e 203, 676-685.\u003c/li\u003e\n\u003cli\u003eBr\u0026ouml;ker K, Sinelnikov E, Gustavus D, Schumacher U, P\u0026ouml;rtner R, Hoffmeister H, L\u0026uuml;th S, Dammermann W (2019) Mass Production of Highly Active NK Cells for Cancer Immunotherapy in a GMP Conform Perfusion Bioreactor. \u003cem\u003eFront Bioeng Biotechnol\u003c/em\u003e 7, 194.\u003c/li\u003e\n\u003cli\u003eSutlu T, Stellan B, Gilljam M, Quezada HC, Nahi H, Gahrton G, Alici E (2010) Clinical-grade, large-scale, feeder-free expansion of highly active human natural killer cells for adoptive immunotherapy using an automated bioreactor. \u003cem\u003eCytotherapy\u003c/em\u003e 12, 1044-1055.\u003c/li\u003e\n\u003cli\u003eKim AMJ, Nemeth MR, Lim SO (2022) 4-1BB: A promising target for cancer immunotherapy. \u003cem\u003eFront Oncol\u003c/em\u003e 12, 968360.\u003c/li\u003e\n\u003cli\u003eDowell AC, Oldham KA, Bhatt RI, Lee SP, Searle PF (2012) Long-term proliferation of functional human NK cells, with conversion of CD56(dim) NK cells to a CD56 (bright) phenotype, induced by carcinoma cells co-expressing 4-1BBL and IL-12. \u003cem\u003eCancer Immunol Immunother\u003c/em\u003e 61, 615-628.\u003c/li\u003e\n\u003cli\u003eVan Acker HH, Capsomidis A, Smits EL, Van Tendeloo VF (2017) CD56 in the Immune System: More Than a Marker for Cytotoxicity? 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However, their limited half-life poses a challenge for therapy. Although attempts to leverage feeder cells are common, safer methods are needed to mitigate the associated risks.\u003c/p\u003e\n\u003cp\u003eIn our study, an upregulation in the expression of 4-1BBL in Colo-205 cells under extracellular stresses such as hypoxia and cytochalasin D was observed. This enhanced binding to the 4-1BB receptors on NK cells promotes proliferation in NK cells. Elevated CD56 expression of a marker strongly linked to NK cell proliferation in co-culture further supports this process.\u003c/p\u003e\n\u003cp\u003eApplying extracellular stressors, specifically hypoxia and cytochalasin D, to Colo-205 cells successfully tailored feeder cells, significantly enhancing NK cell proliferation.\u003c/p\u003e","manuscriptTitle":"Enhanced NK cell proliferation by stress-induced feeder cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-12 12:48:40","doi":"10.21203/rs.3.rs-3918793/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9dceda73-bb17-4140-90d9-1bacb9ad90fa","owner":[],"postedDate":"February 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-29T11:47:29+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-12 12:48:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3918793","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3918793","identity":"rs-3918793","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}