Supercharged natural killer (NK) cells inhibit melanoma tumor progression and restore endogenous NK cell function in humanized BLT mice

Supercharged natural killer (NK) cells inhibit melanoma tumor progression and restore endogenous NK cell function in humanized BLT mice | 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 Supercharged natural killer (NK) cells inhibit melanoma tumor progression and restore endogenous NK cell function in humanized BLT mice Kawaljit Kaur, Paytsar Topchyan, Anahid Jewett This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6792338/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 We have previously shown that a single infusion of supercharged NK cells (sNK) was able to significantly decrease or eliminate oral, pancreatic, and uterine cancers implanted in hu-BLT mice. In this report, we extended those studies to melanoma tumors to observe whether there were differences in response to sNK cells. Furthermore, we studied the effect of sNK cell therapy on PBMCs, bone marrow, and spleen of hu-BLT mice. Our studies indicated that infusion of super-charged NK cells inhibited melanoma tumor growth in hu-BLT mice. In addition, sNK cells circulated through PBMCs, spleen, and bone marrow, and they were also found within the tumor of hu-BLT mice. Moreover, the sNK cells were responsible for the induction of in vivo differentiation of the melanoma tumors. sNK cell infusion increased percentages of NK cells in PBMCs of hu-BLT mice, and restored cytotoxicity and IFN-γ secretion within the PBMCs, spleen, and bone marrow of melanoma tumor-bearing mice. Overall, these results suggested that sNK cell therapy is efficacious in limiting melanoma tumor growth and should be considered as a cell therapy to stop or eliminate these tumors in patients. In addition, sNK cells, by increasing the levels and function of autologous NK cells, should be able to restore the inactivated function of NK cells by the tumor cells in cancer patients. NK cells supercharged NK cells cytotoxicity melanoma IFN-γ humanized-BLT mice cancer stem cells (CSC) immunotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Melanoma cases are increasing in the United States and worldwide, and it is the fifth common cancer diagnosed in the United States, representing 3% of skin cancers and 65% of skin cancer-related deaths [ 1 , 2 ]. The primary treatment approach for melanoma is surgery, but chemotherapy shows prognostic benefits for melanoma patients [ 2 ]. Immunotherapies such as immune checkpoint inhibitors or IL-2 activated T cells have recently shown an increased overall survival of melanoma patients [ 3 – 5 ]. Cancer stem cells (CSCs) play a crucial role in the highly tumorigenic and chemoresistant nature of melanoma [ 6 , 7 ]. Melanoma-related malignant lesions originate from rapid multiplication of skin cancer due to mutations/genetic defects caused by unrepaired DNA damage of skin caused by ultraviolet radiation of sunlight or tanning beds [ 6 ]. Natural killer (NK) cells play crucial roles in cancer inhibition due to their effector functions, including direct cytotoxicity against cancers, antibody-dependent cellular cytotoxicity (ADCC), and indirect regulation of other immune effectors’ function through NK cell-mediated secretion of inflammatory cytokines and chemokines [ 8 – 10 ]. NK cells were found to recognize and target CSCs that express lower surface expression of MHC-class I, CD54, and PD-L1 (B7H1) receptors and higher expression of CD44 [ 11 – 14 ]. In our previous studies, we demonstrated a great correlation between the differentiation stage of the tumors and their susceptibility to primary NK cell-mediated cytotoxicity [ 12 , 15 , 16 ]. The healthy skin immune cell population consists of NK cells with cytotoxic function against stressed or infected melanoma-like cells [ 17 , 18 ]. Studies have shown that the presence of skin fibroblasts can differentiate purified CD56 bright CD16 dim NK cells into CD56 dim NK cells having characteristics and phenotype similar to peripheral blood CD56 dim NK cells [ 19 ]. Decreased NK cell function was found to be associated with poor prognosis of cancer patients [ 20 – 49 ]. The efficacy of NK cells as a cancer therapeutic in solid tumors is very well established [ 50 – 55 ]. Higher surface expression levels of NKG2D ligands MICA/B were found in melanoma cell lines, and during melanoma cells' interaction with NK cells, this ligand interacts with their receptors on NK cells, leading to NK cell activation [ 56 , 57 ]. Ligands for NK cell receptors Nkp44, Nkp46, and Nkp30 were also found to be expressed on melanoma cells [ 58 , 59 ]. Currently, several ongoing studies have shown the efficacy of NK cell-based therapies for melanoma. In this study, we used humanized-BLT (hu-BLT) melanoma tumor-bearing mice injected with supercharged NK (sNK) cells. We found significantly reduced tumor mass and increased survival of mice with sNK cell treatment in comparison to untreated tumor-bearing mice. Treatment with sNK cells resulted in increased percentages of NK cells, increased IFN-γ secretion, and heightened cytotoxic function of immune cells in the peripheral blood-derived mononuclear cells (PBMCs), spleen, and bone marrow of tumor-bearing mice in comparison to tumor-bearing mice. IFN-γ secretion and cytotoxic function of immune cells were also increased in other tissues of tumor-bearing mice infused with sNK cells in comparison to tumor-alone bearing mice. Materials and Methods Cell lines, reagents, and antibodies RPMI 1640 (Gibco, ThermoFisher, CA) supplemented with 10% Fetal Bovine Serum (FBS) (Gemini Bio-Products, CA) was used for the cultures of human NK cells and immune cells of hu-BLT mice. Recombinant IL-2 (rhIL-2) was obtained from NIH-BRB. Flow cytometry and other antibodies used in the study were obtained from BioLegend (San Diego, CA). Human melanoma A375 cells were donated by Dr. Toru Hiraga (Department of Histology and Cell Biology, Matsumoto Dental University, Japan) [ 60 ] and were cultured in DMEM (Life Technologies) supplemented with 10% FBS and 2% antibiotic-antimycotic (Gemini Bio-Products). Oral squamous carcinoma stem cells (OSCSCs) were isolated from patients with tongue tumors at UCLA [ 61 – 64 ]. OSCSCs were cultured in RPMI 1640 medium supplemented with 10% FBS, 2% Antibiotic/Antimycotic Solution (Cytiva, MA), 1.4% Sodium Pyruvate (Gibco, CA, USA), 1.4% MEM Non-Essential Amino Acids (Gibco, CA, USA), and 0.15% sodium bicarbonate. Antibodies that were used for flow cytometry – IgG2, CD16, CD56, CD3, CD45, and MHC-class I were purchased from Biolegend (San Diego, CA, USA). Human NK cells, T cells, and monocyte purification kits were obtained from Stem Cell Technologies (Vancouver, BC, Canada). PKH26 staining kit was purchased from Sigma-Aldrich, St Louis, MO. Human ELISA kits for IFN-γ were purchased from Biolegend (San Diego, CA). Purification of human NK cells and monocytes Written informed consents approved by the UCLA Institutional Review Board (IRB) were obtained, and all procedures were approved by the UCLA-IRB. NK cells and monocytes were negatively selected and isolated from PBMCs using the EasySep® Human NK cell enrichment kit and monocyte isolation kit, respectively, purchased from Stem Cell Technologies (Vancouver, BC, Canada). Isolated NK cells and monocytes were stained with anti-CD16 and anti-CD14 antibodies, respectively, to measure the cell purity using flow cytometric analysis. Probiotic bacteria AJ2 sonication Gram-positive probiotic bacteria: AJ2 (Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei) were weighed and resuspended in RPMI 1640 containing 10% FBS at a concentration of 10 mg/1mL. The bacteria were thoroughly vortexed, then sonicated on ice for 15 seconds, set at a 60% amplitude. Sonicated samples were then incubated for 30 seconds on ice. After every five pulses, a sample was taken to observe under the microscope until at least 80 percent of the cell walls were lysed. It was determined that approximately 20 rounds of sonication/incubation on ice were conducted to achieve complete sonication. Finally, the sonicated sAJ2 samples were aliquoted and stored in a -80 ○ C freezer. Generation of osteoclasts and expansion of human NK cells Purified monocytes from human peripheral blood were cultured in alpha-MEM medium containing M-CSF (25 ng/mL) and RANKL (25 ng/mL) for 21 days, or otherwise specified. The medium was refreshed every 3 days with fresh alpha-MEM containing M-CSF and RANKL. Human purified NK cells were activated with rh-IL-2 (1000 U/ml) and anti-CD16mAb (3 µg/ml) for 18–20 hours before they were co-cultured with osteoclasts and sonicated AJ2 for NK cells expansion. The medium was refreshed every 3 days with RMPI containing rh-IL-2 (1500 U/ml). Analysis of melanoma growth in humanized-BLT mice Animal research was performed under the written approval of the UCLA Animal Research Committee (ARC). Humanized-BLT (hu-BLT; human bone marrow/liver/thymus) mice were prepared as previously described [ 65 , 66 ]. For melanoma i n vivo growth, A375 human melanoma cells (1 x 10 6 ) were mixed 1:1 with HC Matrigel (Corning, Corning, NY) and injected subcutaneously into the right flank of hu-BLT mice as described previously [ 66 , 67 ]. Tumor growth was measured biweekly with a caliper. sNK cells therapeutic group received four weekly tail vein injections of 2 x 10 6 sNK cells. Mice were euthanized when signs of morbidity were evident. Melanoma tumor, bone marrow, spleen, and peripheral blood were harvested from mice at the end of the experiment or when the tumor size reached 2cm in diameter. Cell dissociation and cell culture of tissues from hu-BLT mice The melanoma tumor harvested from hu-BLT mice were immediately cut into 1 mm 3 pieces and placed into a digestion buffer containing 1 mg/ml collagenase IV, 10 U/ml DNAse I, and 1% bovine serum albumin (BSA) in DMEM media, and incubated for 20 minutes at 37 ˚ C oven on a 150 rpm shaker. After digestion, the sample was filtered through a 40 mm cell strainer and centrifuged at 1500 rpm for 10 minutes at 4˚C. The pellet was resuspended in DMEM media, and cells were counted. To obtain single-cell suspensions from BM, femurs were cut from both ends and flushed from one end to the other using RPMI media, BM cells were filtered through a 40 µm cell strainer. To obtain single-cell suspensions from the spleen, the spleen was smashed until no big piece was left, and the sample was filtered through a 40 µm cell strainer and centrifuged at 1500 rpm for 5 minutes at 4˚C. The pellet was re-suspended in ACK buffer to remove the red blood cells for 2–5 minutes, followed by re-suspension in RMPI media and centrifuged at 1500 rpm for 5 minutes at 4˚C. Peripheral blood mononuclear cells (PBMCs) were isolated using ficoll-hypaque centrifugation of heparinized blood specimens. The buffy coat containing PBMCs was harvested, washed, and resuspended in RPMI 1640 medium. Purification of NK cells and CD3 + T cells from hu-BLT mice NK cells and CD3 + T cells from hu-BLT mice splenocytes were isolated using the human CD56 + selection kit and human CD3 + T selection kit, respectively (Stem Cells Technologies, Canada). Enzyme-linked immunosorbent assays (ELISAs) and multiplex cytokine assay The assay was conducted as described in the manufacturer’s protocol. The plates were read in a microplate reader, at 450nm to obtain absorbance values (Biolegend, ELISA manual). To analyze and obtain the cytokine and chemokine concentration, a standard curve was generated by either two or three-fold dilution of recombinant cytokines provided by the manufacturer. Multiplex assay was conducted as described in the manufacturer’s protocol for each specified kit. Analysis was performed using a Luminex multiplex instrument (MAGPIX, Millipore, Billerica, MA), and data was analyzed using the proprietary software (xPONENT 4.2, Millipore, Billerica, MA). Surface Staining assays Staining was performed by labeling the cells with antibodies as described previously [ 68 – 70 ]. Briefly, the cells were washed twice with ice-cold PBS/1% BSA. Predetermined optimal concentrations of specific human flow cytometric antibodies were added to 1 x 10 4 cells in 50 µl of cold-PBS/1%BSA and cells were incubated on ice for 30 min. Thereafter, cells were washed in cold PBS/1%BSA and brought to 500 µl with PBS/1%BSA. Flow cytometry analysis was performed using a Beckman Coulter Epics XL cytometer (Brea, CA), and results were analyzed in FlowJo vX software (Ashland, OR). 51 Cr release cytotoxicity assay The 51 Cr release assay was performed as described previously [ 71 ]. We used OSCSCs as target cells to assess NK cell-mediated cytotoxicity because these cells are the most susceptible cells to NK cell-mediated cytotoxicity [ 41 ]. Briefly, different numbers of effector cells were incubated with 51 Cr–labeled target cells. After a 4-hour incubation period the supernatants were harvested from each sample and counted for released radioactivity using the gamma counter. The percentage specific cytotoxicity was calculated using the following formula: Lytic unit 30/10 6 is calculated by using the inverse of the number of effector cells needed to lyse 30% of the tumor target cells X100. Statistical analysis An unpaired, two-tailed student t-test was performed for the statistical analysis. One-way ANOVA using Prism-10 software was used to compare different groups. (n) denotes the number of mice used for each condition in the experiment. The following symbols represent the levels of statistical significance within each analysis: ***(p-value < 0.001), **(p-value 0.001–0.01), *(p-value 0.01–0.05). Results Infusion of super-charged NK cells inhibited melanoma tumor growth in hu-BLT mice Hu-BLT mice were generated, and human melanoma tumor cells were implanted subcutaneously in mice as described in the Materials and Methods section. In vivo tumor growth was monitored weekly basis, and we observed a lower tumor growth rate in the mice infused with sNK cells (Fig. 1 A). Tumor-bearing mice infused with sNK cells did not exhibit morbidity and were able to climb in the cage, whereas untreated tumor-bearing mice became morbid and had complications in climbing and running. The tumor inhibiting potential of sNK cells was also validated by the small tumor size in the therapeutic group, post-sacrifice (Fig. 1 B). When we dissociated the tumor, a higher number of tumor cells (Fig. 1 C) and increased ex vivo tumor growth (Fig. 1 D) were observed in the tumor resected from untreated mice compared to sNK cell-treated group. sNK cells circulated through PBMCs, spleen, and bone marrow and were found within the tumor of hu-BLT mice, and were responsible for the induction of in vivo differentiation of the melanoma tumors In order to track sNK cells in mouse tissues, sNK cells were labeled with PKH dye before infusion. Post-sacrifice, we detected sNK cells in peripheral blood (17.2%), spleen (29.6%), bone marrow (4.64%), and tumor (2.12%) (Fig. 2 A). When tumor-infiltrating human NK and T cells were assessed, increased percentages of NK and NKT cells were found to be infiltrating in tumors of tumor-bearing mice infused with sNK cells when compared to untreated tumor-bearing mice (Fig. 2 B). Tumors were cultured ex vivo for 7 days, increased percentages of human CD45 + immune cells were seen in both untreated and IL-2 treated tumors from tumor-bearing mice with sNK infusion compared to untreated tumor-bearing mice (Fig. 2 C). We also have examined the surface expression of differentiation antigens MHC-class I on tumors, and found higher MHC-class I surface expression on tumors obtained from sNK cells infused tumor-bearing mice as compared to untreated tumor-bearing mice (Fig. 2 D). sNK cell infusion increased percentages of NK cells and restored IFN-γ secretion within the tissue compartments of melanoma tumor-bearing mice Higher NK cell percentages in PBMCs (Fig. 3 A) and spleen (Fig. 5 A) were seen in tumor-bearing hu-BLT mice infused with sNK cells. There was no change or a slight decrease in percentages of T cells in peripheral blood (Fig. 3 B) and spleen (Fig. 5 B). A different profile of NK and T cell percentages was seen in the BM of tumor-bearing mice injected with sNK. There was a slight decrease in NK cell percentages, whereas a slight increase in T cell percentages was observed (Figs. 4 A- 4 B) in tumor-bearing mice injected with sNK cells. Increased secretion of IFN-γ was observed in peripheral blood-derived serum (Table 1 ), PBMCs (Figs. 3 C and S1A, and Table 2 ), BM (Figs. 4 C and 4 E), and spleen (Figs. 5 C and 5 E, and Table 3 ) of tumor-bearing mice injected with sNK cells. Increased secretion of other factors, except IL-10, was seen in peripheral blood-derived serum (Table 1 ). PBMCs expressed increased secretion of factors except VEGF (Table 2 ). Spleen from tumor-bearing mice injected with sNK demonstrated increased secretion of factors determined in this study (Table 3 ). Table 1 Infusion of sNK cells restored secretion of IFN-γ and other factors in the peripheral blood-derived serum of melanoma tumor-bearing hu-BLT mice Serum IFN-γ TNF-α IL-10 IL-12 IL-17A IL-1b IL-2 Tumor 5.5 +/- 3 7 +/- 1.4 33.5 +/- 16 3 +/- 1.4 6 +/- 0.1 2 +/- 1.1 5 +/- 2.1 Tumor + sNK 60 26 17 24 48 16 16 Serum GMCSF MIP-1b ITAC Fractalkine MIP-3A Tumor 291.5 +/- 168 6.5 +/- 0.7 16 +/- 7 109 +- 12 4 +/- 1.1 Tumor + sNK 328 33 32 320 27 Table 2 Infusion of sNK cells restored secretion of IFN-γ and other factors in the spleen of melanoma tumor-bearing hu-BLT mice PBMCs IFN-γ TNF-α IL-6 IFN-a VEGF GMCSF IP-10 Eotaxin Fractalkine Tumor 0.9 +/- 0.7 2.5 +/- 1.6 5 +/- 3.3 10 +/- 5.8 57 +/- 10 0.5 +/- 0.5 3.2 +/- 0.1 3.8 +/- 1 14 +/- 5 Tumor + sNK 2 +/- 0.7 4.1 +/- 3 7.7 +/- 2.3 24 +/- 2 33.6 +/- 12 3.1 +/- 1.9 6 +/- 2.5 4.3 +/- 0.6 22 +/- 7 Table 3 Spleen IFN-γ TNF-α IL-6 IL-10 GMCSF MCP-1 MIP-1a MIP-1b MDC IP-10 Tumor 3 +/- 2 3.7 +/- 1.2 1.9 +/- 1 2.5 +/- 0.6 6.15 +/- 2.8 5.3 +/- 3.5 202 +/- 165 63 +/- 17 211 +/- 91 14.7 +/- 4.5 Tumor + sNK 6.5 +/- 2.5 6.9 +/- 1.8 4.9 +/- 1.1 24 +/- 18 12.2 +/- 0.1 14.1 +/- 2.3 805 +/- 296 118 +/- 48 405 +/- 147 34 +- 21 Increased NK cell-mediated cytotoxicity was observed in PBMCs (Figs. 3 D and S1B) and BM-derived immune cells (Figs. 4 D and 4 F) from tumor-bearing mice infused with sNK cells. Different NK cell-mediated cytotoxicity profile was seen in the spleen; sNK cells infused groups showed decreased levels of NK cell-mediated cytotoxicity compared to the untreated tumor-bearing group (Figs. 5 D and 5 F). sNK cells-infused group also expressed an increased level of IFN-γ was observed in spleen-derived NK (Fig. 6 A) and T cells (Fig. S2), and also expressed increased NK cell-mediated activity in spleen-derived NK cells (Fig. 6 B). Overall, restored immune function was observed with sNK cells therapy in melanoma tumor-bearing mice. Discussion We have previously shown that a single infusion of supercharged NK cells was able to significantly decrease or eliminate oral, pancreatic, and uterine cancers in hu-BLT mice [ 72 – 75 ]. In this report, we extended those studies to melanoma tumors to see if there were any differences in response to sNK cells. In addition, we studied the effect of sNK cell therapy, not only within the tumor microenvironment, but also the effect on PBMCs, bone marrow, and spleen. Infusion of sNK cells resulted in increased numbers of circulating PKH-stained sNK cells in PBMCs, spleen, bone marrow, and the tumor (Fig. 2 A). Interestingly, the levels of sNK cells were the highest in the spleen, then PBMCs, and the least in bone marrow. There were increased levels of sNK cells in the tumor cells, although the percentages were lower in comparison to the other tissues (Fig. 2 A). Infusion of sNK cells significantly decreased the size and the weight of the tumor and delayed the growth of the tumors (Figs. 1 A- 1 C). When single cells were prepared from the tumors and equal numbers of tumors from both groups were cultured for several days, much less tumor growth was seen in the tumor cells from tumor-implanted mice treated with sNK cells as compared to the untreated group. We then focused on the type of immune cells circulating in the tumor cells. After single-cell preparation of the tumors, the levels of CD3 and CD56 subpopulations were determined in the tumor microenvironment. There was an increased percentage of CD3-CD56 + NK cells in addition to an increase in total CD3 + T cells and CD3 + CD56 + NKT cells within the tumor cells (Fig. 2 B). We then cultured the tumor cells with and without IL-2 treatment and measured the circulating CD45 + immune cells. The proportion of CD45 + immune cells was higher in sNK-infused tumor-implanted BLT mice as compared to tumor alone-implanted mice, and the addition of IL-2 further increased the levels of CD45 + immune cells within the tumor cultures. We have previously shown that sNK-mediated increase in NK cells and subsequent induction of IFN-γ increase the levels of MHC-class I on the tumor cells. Accordingly, we observed higher expression of MHC-class I on tumors resected from tumors implanted in hu-BLT mice infused with sNK cells. Treatment of the tumors resected from tumor-implanted hu-BLT mice infused with sNK and treated with IL-2 substantially increased the levels of MHC-class I expression when compared to tumors alone implanted mice (Fig. 2 D). These studies indicated that sNK cells circulate within the tumor and are likely responsible in part for the increased MHC-class I levels on tumors, which we have shown to be part of the four major biomarkers indicating the levels of differentiation of the tumor cells [ 74 – 76 ]. We then assessed the levels of circulating NK cells in PBMCs and found that the levels of NK cells were elevated in tumor-implanted mice infused with sNK cells. Due to the higher variability between mice, we could not show statistical significance, even though on average the levels were much higher in tumor-implanted mice infused with sNK cells as compared to only tumor-implanted mice. This observation is very important since we see the same trend in human patients infused with sNK cells, in which case the percentages of NK cells rise significantly and remain high for greater than 1 year (manuscript submitted). Based on chip analysis in humans, the elevated NK cells are from the recipient (autologous) than the donor's sNK cells. In this study, the donor's sNK cells were from male donors, and the recipient was female; therefore, in the chip assay, we could only see the X chromosome and not the Y chromosome, indicating that the elevated percentages of NK cells were autologous and not allogeneic. We then assessed the levels of IFN-γ secretion and NK cell-mediated cytotoxicity by PBMCs and found that to be significantly higher levels of IFN-γ and cytotoxicity against NK-specific targets (Figs. 3 C- 3 E, and S1). Interestingly, no difference could be seen for the levels of T cells within PBMCs of mice implanted with a tumor and infused with sNK cells as compared to tumor alone implanted mice. In addition, when other cytokines and chemokines were assessed in supernatants recovered from PBMCs, we have not only observed higher IFN-γ and TNF-α release, but also many other cytokines and chemokines were upregulated in tumor mice infused with sNK cells as compared to tumor mice alone (Table 2 ). We have shown previously that the combination of IFN-γ and TNF-α elevated MHC-class I, CD54, and PDL1, whereas it decreased CD44 expression, a profile which was used to distinguish between stem-like cells and their differentiated counterparts [ 76 , 77 ]. Differentiation of tumor cells with IFN-γ and TNF-α slowed tumor growth substantially [ 76 , 77 ]. In addition to PBMCs, we also observed similar patterns of increased cytokine and chemokine release in the serum of tumor-implanted hu-BLT mice that received infusion of sNK cells as compared to tumor alone-implanted mice (Table 1 ). Both IFN-γ and TNF-α release were upregulated in tumor mice receiving sNK cells (Table 1 ). In contrast, the circulating NK cells in bone marrow were on average lower in tumor mice infused with sNK cells (Fig. 4 A). At the moment it is unclear why bone marrow exhibits differently from PBMC and spleen (please see below). However, in accordance with PBMCs and spleen, the bone marrow cells exhibited significantly higher secretion of IFN-γ and mediated higher NK cell-mediated cytotoxicity from tumor mice infused with sNK cells as compared to tumor alone mice (Figs. 3 – 5 ). As for the spleen, it exhibited higher percentages of NK cells in tumor mice infused with sNK cells, and the cells mediated increased secretion of IFN-γ while mediating lower cytotoxicity as compared to tumor alone implanted mice (Fig. 5 ). Similarly, we saw higher secretion of cytokines and chemokines from splenocytes when assessed by multiplex assay (Table 3 ). We then sorted the NK cells and T cells from the spleens of the mice and performed functional assays. We observed significantly increased secretion of INF-γ and augmented cytotoxicity by the NK cells sorted from tumor mice infused with the sNK cells (Fig. 6 ). Likewise, sorted T cells were also able to increase the levels of IFN-γ from tumor mice infused with sNK cells, although due to high variability between mice, we could not achieve statistical significance (Fig. S2). We have shown previously that sNK cells preferentially expand CD8 + T cells and increase their functional capabilities [ 47 , 75 ]. Overall, these results suggested that sNK cell therapy should be efficacious in melanoma and should be considered as a cell therapy to stop or eliminate these tumors. In addition, sNK cells, by increasing the levels and function of autologous NK cells, should be able to restore the inactivated NK cells by the tumor cells (Figs. 3 – 5 , and Tables 1 – 3 )[ 47 , 72 – 74 ]. Abbreviations NK cells: Natural killer cells sNK cells: Supercharged NK cells CSCs: Cancer-stem-like-cells Hu-BLT: Humanized-bone marrow/liver/thymus IFN-γ : Interferon-gamma IL-2: Interleukin 2 OCs: Osteoclasts PBMCs: Peripheral blood-derived mononuclear cells OSCSCs: Oral squamous cancer stem-like cells ELISAs: Enzyme-Linked Immunosorbent Assays Cr: Chromium Declarations Ethical approval and consent to participate : Protocols allowing the collection and use of human specimens for this study were approved by the UCLA Office of the Human Research Protection Program (IRB# 20-1659). Written informed consents, approved by the UCLA Institutional Review Board ( IRB#11-000781), were obtained from healthy individuals and ovarian cancer patients, and the UCLA-IRB approved all procedures. Animal research was performed under the written approval of the UCLA Animal Research Committee (ARC) in accordance with all federal, state, and local guidelines. Consent for publication : The authors provide consent for publication to be published in the Cancer Immunology, Immunotherapy. Availability of data and material: The data presented in the study is included in the main and supplementary files of this manuscript. Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential competing interest. Author Contributions: KK generated and analyzed data, wrote, reviewed, and edited the manuscript. PT generated data. 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Ann Surg Oncol, 2016. 23 Suppl 2 : p. S257-65. Baskic, D., et al., Suppression of natural killer-cell and dendritic-cell apoptotic tumoricidal activity in patients with head and neck cancer. Head Neck, 2013. 35 (3): p. 388-98. Tseng, H.C., N. Cacalano, and A. Jewett, Split anergized Natural Killer cells halt inflammation by inducing stem cell differentiation, resistance to NK cell cytotoxicity and prevention of cytokine and chemokine secretion. Oncotarget, 2015. 6 (11): p. 8947-59. Mickel, R.A., et al., Natural killer cell cytotoxicity in the peripheral blood, cervical lymph nodes, and tumor of head and neck cancer patients. Cancer Res, 1988. 48 (17): p. 5017-22. Ghoneum, M., G. Gill, and L. Perry, Natural killer cell activity in patients with carcinoma of the larynx and hypopharynx. Laryngoscope, 1986. 96 (11): p. 1300. Tartter, P.I., et al., The prognostic significance of natural killer cytotoxicity in patients with colorectal cancer. Arch Surg, 1987. 122 (11): p. 1264-8. Izawa, S., et al., H2O2 production within tumor microenvironment inversely correlated with infiltration of CD56dim NK cells in gastric and esophageal cancer: possible mechanisms of NK cell dysfunction. Cancer Immunology, Immunotherapy, 2011. 60 (12): p. 1801-1810. Nolibe, D. and M.F. Poupon, Enhancement of pulmonary metastases induced by decreased lung natural killer cell activity. J Natl Cancer Inst, 1986. 77 (1): p. 99-103. Kaur, K., et al., Osteoclast-expanded super-charged NK-cells preferentially select and expand CD8+ T cells. Sci Rep, 2020. 10 (1): p. 20363. Kaur, K., et al., Defective NK cell expansion, cytotoxicity, and lack of ability to differentiate tumors from a pancreatic cancer patient in a long term follow-up: implication in the progression of cancer. Cancer Immunol Immunother, 2022. 71 (5): p. 1033-1047. Kaur, K., et al., Novel Strategy to Expand Super-Charged NK Cells with Significant Potential to Lyse and Differentiate Cancer Stem Cells: Differences in NK Expansion and Function between Healthy and Cancer Patients. Front Immunol, 2017. 8 : p. 297. Ruggeri, L., et al., Effectiveness of Donor Natural Killer Cell Alloreactivity in Mismatched Hematopoietic Transplants. Science, 2002. 295 (5562): p. 2097-2100. Venstrom, J.M., et al., HLA-C-dependent prevention of leukemia relapse by donor activating KIR2DS1. N Engl J Med, 2012. 367 (9): p. 805-16. Iliopoulou, E.G., et al., A phase I trial of adoptive transfer of allogeneic natural killer cells in patients with advanced non-small cell lung cancer. Cancer Immunol Immunother, 2010. 59 (12): p. 1781-9. Miller, J.S., et al., Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood, 2005. 105 (8): p. 3051-7. Re, F., et al., Killer cell Ig-like receptors ligand-mismatched, alloreactive natural killer cells lyse primary solid tumors. Cancer, 2006. 107 (3): p. 640-8. Geller, M.A., et al., A phase II study of allogeneic natural killer cell therapy to treat patients with recurrent ovarian and breast cancer. Cytotherapy, 2011. 13 (1): p. 98-107. Casado, J.G., et al., Expression of adhesion molecules and ligands for activating and costimulatory receptors involved in cell-mediated cytotoxicity in a large panel of human melanoma cell lines. Cancer Immunol Immunother, 2009. 58 (9): p. 1517-26. Vetter, C.S., et al., Expression of stress-induced MHC class I related chain molecules on human melanoma. J Invest Dermatol, 2002. 118 (4): p. 600-5. Cagnano, E., et al., Expression of ligands to NKp46 in benign and malignant melanocytes. J Invest Dermatol, 2008. 128 (4): p. 972-9. Byrd, A., et al., Expression analysis of the ligands for the Natural Killer cell receptors NKp30 and NKp44. PLoS One, 2007. 2 (12): p. e1339. Hiraga, T., S. Ito, and H. Nakamura, Cancer stem-like cell marker CD44 promotes bone metastases by enhancing tumorigenicity, cell motility, and hyaluronan production. Cancer Res, 2013. 73 (13): p. 4112-22. Tseng, H.C., et al., Increased lysis of stem cells but not their differentiated cells by natural killer cells; de-differentiation or reprogramming activates NK cells. PLoS One, 2010. 5 (7): p. e11590. Tseng, H.C., et al., Induction of Split Anergy Conditions Natural Killer Cells to Promote Differentiation of Stem Cells through Cell-Cell Contact and Secreted Factors. Front Immunol, 2014. 5 : p. 269. Tseng, H.C., et al., Differential Targeting of Stem Cells and Differentiated Glioblastomas by NK Cells. J Cancer, 2015. 6 (9): p. 866-76. Bui, V.T., et al., Augmented IFN-γ and TNF-α Induced by Probiotic Bacteria in NK Cells Mediate Differentiation of Stem-Like Tumors Leading to Inhibition of Tumor Growth and Reduction in Inflammatory Cytokine Release; Regulation by IL-10. Frontiers in Immunology, 2015. 6 . Shimizu, S., et al., A highly efficient short hairpin RNA potently down-regulates CCR5 expression in systemic lymphoid organs in the hu-BLT mouse model. Blood, 2010. 115 (8): p. 1534-44. Vatakis, D.N., et al., Antitumor activity from antigen-specific CD8 T cells generated in vivo from genetically engineered human hematopoietic stem cells. Proc Natl Acad Sci U S A, 2011. 108 (51): p. E1408-16. Vatakis, D.N., et al., Using the BLT humanized mouse as a stem cell based gene therapy tumor model. J Vis Exp, 2012(70): p. e4181. Jewett, A., M. Cavalcanti, and B. Bonavida, Pivotal role of endogenous TNF-alpha in the induction of functional inactivation and apoptosis in NK cells. J Immunol, 1997. 159 (10): p. 4815-22. Jewett, A. and B. Bonavida, Interferon-alpha activates cytotoxic function but inhibits interleukin-2-mediated proliferation and tumor necrosis factor-alpha secretion by immature human natural killer cells. J Clin Immunol, 1995. 15 (1): p. 35-44. Jewett, A. and B. Bonavida, Target-induced inactivation and cell death by apoptosis in a subset of human NK cells. J Immunol, 1996. 156 (3): p. 907-15. Jewett, A., et al., Cytokine dependent inverse regulation of CD54 (ICAM1) and major histocompatibility complex class I antigens by nuclear factor kappaB in HEp2 tumor cell line: effect on the function of natural killer cells. Hum Immunol, 2003. 64 (5): p. 505-20. Kaur, K. and A. Jewett, Supercharged NK Cell-Based Immuotherapy in Humanized Bone Marrow Liver and Thymus (Hu-BLT) Mice Model of Oral, Pancreatic, Glioblastoma, Hepatic, Melanoma and Ovarian Cancers. Crit Rev Immunol, 2023. 43 (2): p. 13-25. Kaur, K., et al., Sequential therapy with supercharged NK cells with either chemotherapy drug cisplatin or anti-PD-1 antibody decreases the tumor size and significantly enhances the NK function in Hu-BLT mice. Front Immunol, 2023. 14 : p. 1132807. Kaur, K., et al., Probiotic-Treated Super-Charged NK Cells Efficiently Clear Poorly Differentiated Pancreatic Tumors in Hu-BLT Mice. Cancers (Basel), 2019. 12 (1). Kaur, K., et al., Super-charged NK cells inhibit growth and progression of stem-like/poorly differentiated oral tumors in vivo in humanized BLT mice; effect on tumor differentiation and response to chemotherapeutic drugs. Oncoimmunology, 2018. 7 (5): p. e1426518. Bui, V.T., et al., Augmented IFN-γ and TNF-α Induced by Probiotic Bacteria in NK Cells Mediate Differentiation of Stem-Like Tumors Leading to Inhibition of Tumor Growth and Reduction in Inflammatory Cytokine Release; Regulation by IL-10. Front Immunol, 2015. 6 : p. 576. Kaur, K., A.P. Celis, and A. Jewett, Natural Killer Cell-Secreted IFN-γ and TNF-α Mediated Differentiation in Lung Stem-like Tumors, Leading to the Susceptibility of the Tumors to Chemotherapeutic Drugs. Cells, 2025. 14 (2): p. 90. Additional Declarations No competing interests reported. Supplementary Files SupplementaryDatamelanomaCII.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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6792338","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":465944020,"identity":"c45ff910-1505-4c34-9e87-5bff425cdbfc","order_by":0,"name":"Kawaljit Kaur","email":"","orcid":"","institution":"UCLA School of Dentistry","correspondingAuthor":false,"prefix":"","firstName":"Kawaljit","middleName":"","lastName":"Kaur","suffix":""},{"id":465944021,"identity":"3bda8448-e63e-4da4-8b9b-feda5fa008ef","order_by":1,"name":"Paytsar Topchyan","email":"","orcid":"","institution":"UCLA School of Dentistry","correspondingAuthor":false,"prefix":"","firstName":"Paytsar","middleName":"","lastName":"Topchyan","suffix":""},{"id":465944022,"identity":"cdbceecd-714a-4616-9e74-e446a81342b0","order_by":2,"name":"Anahid Jewett","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYFCCA2BSDogNIALsIIKNkJYEA2OIlgQgxUxQCwNYS2ID0Vr4Gw8fk/j440/62vbmbQ8+/mBI7G9mfsDwoewwTi0SB46lSc5IMMjdduZYueGMBIbEGYfZDBhnnMOtheHAGbPbPCAtN3LMpHmAWjYwMxgw87bh1iJ/4Py3238SDNLN7r8xk/4D1sL+gfkvHi0GB86w3QZ6P8HsBo+ZNANYC48BMyMeLYYHjpn/7EkzNtx2Jq1MsidNwnjGYZ6Cgz3n0nFqkbtx+LHBDxs5ebPjh7dJ/LCxke1vb9/44EeZNW7vSxxA5YLJA5jqkAB/A17pUTAKRsEoGAUMDAAYrlpF/XYkmgAAAABJRU5ErkJggg==","orcid":"","institution":"UCLA School of Dentistry","correspondingAuthor":true,"prefix":"","firstName":"Anahid","middleName":"","lastName":"Jewett","suffix":""}],"badges":[],"createdAt":"2025-05-31 17:38:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6792338/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6792338/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84201586,"identity":"c09248dc-1e3e-4c23-8a05-9d157c53f9bf","added_by":"auto","created_at":"2025-06-09 08:31:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":191081,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInfusion of sNK cells inhibited tumor growth in melanoma tumor-bearing hu-BLT mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHu-BLT mice were injected subcutaneously with 1 x 10\u003csup\u003e6\u003c/sup\u003e of human A375 melanoma cells in the right flank. One week after tumor implantation, the therapeutic group received 2 x 10\u003csup\u003e6\u003c/sup\u003e sNK cells weekly dose (total four doses) via tail vein injection. In vivo tumor size was monitored on the days as shown in the figure \u003cstrong\u003e(A)\u003c/strong\u003e. At the end of the experiment, mice were sacrificed, and the pictures of tumors were weighed (n=3) \u003cstrong\u003e(B)\u003c/strong\u003e. Tumor samples were dissociated, and tumor cells were counted (n=3) \u003cstrong\u003e(C)\u003c/strong\u003e, and ex vivo tumor cultures were performed \u003cstrong\u003e(D)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6792338/v1/e80d5f53556a0120ee1c3dfd.png"},{"id":84201590,"identity":"126d6394-34d0-4797-9226-ff8d0672b1c4","added_by":"auto","created_at":"2025-06-09 08:31:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":273032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInfused sNK cells distributed to tissues and tumors, resulting in vivo melanoma tumor differentiation in hu-BLT mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHu-BLT were implanted with melanoma tumors and injected with NK cells as described in Figure 1. sNK were PKH-labeled before infusion. At the end of the experiment, mice were sacrificed, and reconstitution of sNK cells was determined in PBMCs, spleen, BM, and tumors using flow cytometry \u003cstrong\u003e(A)\u003c/strong\u003e. Dissected tumor samples from hu-BLT mice were dissociated, and surface expression of human CD3 and CD56 was determined using antibody staining followed by flow cytometric analysis \u003cstrong\u003e(B)\u003c/strong\u003e. Single cell suspensions (dissociated tumors) of the tumor were cultured untreated or treated with IL-2 (1000 U/ml) (3 x 10\u003csup\u003e6\u003c/sup\u003e cells/ml) from each group for 7 days. The percentages of infiltrating hu-CD45\u003csup\u003e+\u003c/sup\u003e immune cells within the non-attached cells \u003cstrong\u003e(C) \u003c/strong\u003eand MHC-class I on attached cells \u003cstrong\u003e(D)\u003c/strong\u003e at day 7 of culture were determined using antibody staining followed by flow cytometric analysis.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6792338/v1/4ae39872604739868387326a.png"},{"id":84201591,"identity":"580388ca-92ab-4c4a-81be-ec8a2d61bb1a","added_by":"auto","created_at":"2025-06-09 08:31:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":130354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInfusion of sNK cells increased percentages of endogenous NK cells, and restored IFN-γ secretion and NK cell-mediated cytotoxic function in the peripheral blood of melanoma tumor-bearing hu-BLT mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHu-BLT were implanted with melanoma tumors and injected with NK cells as described in Figure 1. Following sacrifice, peripheral blood was collected, single cell suspensions were prepared, and surface expression of human CD16+CD56+ (n=3) \u003cstrong\u003e(A) \u003c/strong\u003eand CD3 (n=3) \u003cstrong\u003e(B)\u003c/strong\u003e was determined using antibody staining followed by flow cytometric analysis. Peripheral blood-derived single cell suspensions were treated with IL-2 (1000 U/ml) and were cultured for 7 days, after which the supernatants were harvested and the levels of IFN-γ were determined using specific ELISAs \u003cstrong\u003e(C)\u003c/strong\u003e. Peripheral blood-derived single cell suspensions were treated with IL-2 (1000 U/ml) and were cultured for 7 days, and cytotoxicity assays were performed using a standard 4-hour \u003csup\u003e51\u003c/sup\u003eCr release assay against OSCSCs, and the LU 30/10\u003csup\u003e6 \u003c/sup\u003ecells were determined using an inverse number of cells required to lyse 30% of OSCSCs x100 \u003cstrong\u003e(D)\u003c/strong\u003e. One of three representative experiments is shown in Figure C-D.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6792338/v1/d700e08373cd956cdeefacd5.png"},{"id":84202884,"identity":"5b7c008f-22e1-4dc3-b1b8-89cadef4c507","added_by":"auto","created_at":"2025-06-09 08:39:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":198038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInfusion of sNK cells restored IFN-γ secretion and NK cell-mediated cytotoxic function in the bone marrow of melanoma tumor-bearing hu-BLT mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHu-BLT were implanted with melanoma tumors and injected with NK cells as described in Figure 1. Following sacrifice, bone marrow was collected, single cell suspensions were prepared, and surface expression of human CD16+CD56+ (n=3) \u003cstrong\u003e(A) \u003c/strong\u003eand CD3 (n=3) \u003cstrong\u003e(B)\u003c/strong\u003e was determined using antibody staining followed by flow cytometric analysis. Bone marrow-derived single cell suspensions were treated with IL-2 (1000 U/ml) and were cultured for 7 days, after which the supernatants were harvested and the levels of IFN-γ were determined using specific ELISAs (n=4) \u003cstrong\u003e(C)\u003c/strong\u003e. Bone marrow-derived single cell suspensions were treated with IL-2 (1000 U/ml) and were cultured for 7 days, and cytotoxicity assays were performed using a standard 4-hour \u003csup\u003e51\u003c/sup\u003eCr release assay against OSCSCs, and the LU 30/10\u003csup\u003e6 \u003c/sup\u003ecells were determined using an inverse number of cells required to lyse 30% of OSCSCs x100 (n=4) \u003cstrong\u003e(D)\u003c/strong\u003e. One of three representative experiments is shown in Figure E-F.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6792338/v1/57b959e399cef0499697eeae.png"},{"id":84202885,"identity":"55d78767-e5ae-40fd-a256-b8c679b23105","added_by":"auto","created_at":"2025-06-09 08:39:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":196574,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInfusion of sNK cells increased percentages of endogenous NK cells, and restored IFN-γ secretion and NK cell-mediated cytotoxic function in the spleen of melanoma tumor-bearing hu-BLT mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHu-BLT were implanted with melanoma tumors and injected with NK cells as described in Figure 1. Following sacrifice, spleen was collected, single cell suspensions were prepared, and surface expression of human CD16+CD56+ (n=4) \u003cstrong\u003e(A) \u003c/strong\u003eand CD3 (n=4) \u003cstrong\u003e(B)\u003c/strong\u003e was determined using antibody staining followed by flow cytometric analysis. Spleen-derived single cell suspensions were treated with IL-2 (1000 U/ml) and were cultured for 7 days, after which the supernatants were harvested and the levels of IFN-γ were determined using specific ELISAs (n=4) \u003cstrong\u003e(C)\u003c/strong\u003e. Spleen-derived single cell suspensions were treated with IL-2 (1000 U/ml) and were cultured for 7 days, and cytotoxicity assays were performed using a standard 4-hour \u003csup\u003e51\u003c/sup\u003eCr release assay against OSCSCs, and the LU 30/10\u003csup\u003e6 \u003c/sup\u003ecells were determined using an inverse number of cells required to lyse 30% of OSCSCs x100 (n=3) \u003cstrong\u003e(D)\u003c/strong\u003e. One of three representative experiments is shown in Figure E-F.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6792338/v1/df37762a7603b22cba2375ed.png"},{"id":84201593,"identity":"c0d404c0-87d5-4e52-93cd-cc06b101362b","added_by":"auto","created_at":"2025-06-09 08:31:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":71958,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInfusion of sNK cells restored IFN-γ secretion and NK cell-mediated cytotoxic function in the spleen-derived NK cells of melanoma tumor-bearing hu-BLT mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHu-BLT were implanted with melanoma tumors and injected with NK cells as described in Figure 1. Following sacrifice, the spleen was collected, single cell suspensions were prepared, and NK cells were purified. Spleen-derived NK cells were treated with IL-2 (1000 U/ml) and were cultured for 7 days, after which the supernatants were harvested and the levels of IFN-γ were determined using specific ELISAs (n=4) \u003cstrong\u003e(A)\u003c/strong\u003e. Spleen-derived NK cells were treated with IL-2 (1000 U/ml) and were cultured for 7 days, and cytotoxicity assays were performed using a standard 4-hour \u003csup\u003e51\u003c/sup\u003eCr release assay against OSCSCs, and the LU 30/10\u003csup\u003e6 \u003c/sup\u003ecells were determined using an inverse number of cells required to lyse 30% of OSCSCs x100 (n=4) \u003cstrong\u003e(B)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6792338/v1/e742ff38e97cfacb499eade7.png"},{"id":84255199,"identity":"19507deb-cf9b-42f4-a66d-f01e3cd38e48","added_by":"auto","created_at":"2025-06-09 19:53:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2605431,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6792338/v1/33966507-92a8-4d56-8cb6-04191f3f30ec.pdf"},{"id":84201588,"identity":"bfcff5b3-321d-4f65-9741-d5dd2803108f","added_by":"auto","created_at":"2025-06-09 08:31:59","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":77848,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryDatamelanomaCII.docx","url":"https://assets-eu.researchsquare.com/files/rs-6792338/v1/cf32c48150f1ccc92bd964a1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Supercharged natural killer (NK) cells inhibit melanoma tumor progression and restore endogenous NK cell function in humanized BLT mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMelanoma cases are increasing in the United States and worldwide, and it is the fifth common cancer diagnosed in the United States, representing 3% of skin cancers and 65% of skin cancer-related deaths [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The primary treatment approach for melanoma is surgery, but chemotherapy shows prognostic benefits for melanoma patients [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Immunotherapies such as immune checkpoint inhibitors or IL-2 activated T cells have recently shown an increased overall survival of melanoma patients [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Cancer stem cells (CSCs) play a crucial role in the highly tumorigenic and chemoresistant nature of melanoma [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Melanoma-related malignant lesions originate from rapid multiplication of skin cancer due to mutations/genetic defects caused by unrepaired DNA damage of skin caused by ultraviolet radiation of sunlight or tanning beds [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNatural killer (NK) cells play crucial roles in cancer inhibition due to their effector functions, including direct cytotoxicity against cancers, antibody-dependent cellular cytotoxicity (ADCC), and indirect regulation of other immune effectors\u0026rsquo; function through NK cell-mediated secretion of inflammatory cytokines and chemokines [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. NK cells were found to recognize and target CSCs that express lower surface expression of MHC-class I, CD54, and PD-L1 (B7H1) receptors and higher expression of CD44 [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In our previous studies, we demonstrated a great correlation between the differentiation stage of the tumors and their susceptibility to primary NK cell-mediated cytotoxicity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe healthy skin immune cell population consists of NK cells with cytotoxic function against stressed or infected melanoma-like cells [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Studies have shown that the presence of skin fibroblasts can differentiate purified CD56\u003csup\u003ebright\u003c/sup\u003eCD16\u003csup\u003edim\u003c/sup\u003e NK cells into CD56\u003csup\u003edim\u003c/sup\u003e NK cells having characteristics and phenotype similar to peripheral blood CD56\u003csup\u003edim\u003c/sup\u003e NK cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Decreased NK cell function was found to be associated with poor prognosis of cancer patients [\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28 CR29 CR30 CR31 CR32 CR33 CR34 CR35 CR36 CR37 CR38 CR39 CR40 CR41 CR42 CR43 CR44 CR45 CR46 CR47 CR48\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The efficacy of NK cells as a cancer therapeutic in solid tumors is very well established [\u003cspan additionalcitationids=\"CR51 CR52 CR53 CR54\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Higher surface expression levels of NKG2D ligands MICA/B were found in melanoma cell lines, and during melanoma cells' interaction with NK cells, this ligand interacts with their receptors on NK cells, leading to NK cell activation [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Ligands for NK cell receptors Nkp44, Nkp46, and Nkp30 were also found to be expressed on melanoma cells [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Currently, several ongoing studies have shown the efficacy of NK cell-based therapies for melanoma.\u003c/p\u003e \u003cp\u003eIn this study, we used humanized-BLT (hu-BLT) melanoma tumor-bearing mice injected with supercharged NK (sNK) cells. We found significantly reduced tumor mass and increased survival of mice with sNK cell treatment in comparison to untreated tumor-bearing mice. Treatment with sNK cells resulted in increased percentages of NK cells, increased IFN-γ secretion, and heightened cytotoxic function of immune cells in the peripheral blood-derived mononuclear cells (PBMCs), spleen, and bone marrow of tumor-bearing mice in comparison to tumor-bearing mice. IFN-γ secretion and cytotoxic function of immune cells were also increased in other tissues of tumor-bearing mice infused with sNK cells in comparison to tumor-alone bearing mice.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines, reagents, and antibodies\u003c/h2\u003e \u003cp\u003eRPMI 1640 (Gibco, ThermoFisher, CA) supplemented with 10% Fetal Bovine Serum (FBS) (Gemini Bio-Products, CA) was used for the cultures of human NK cells and immune cells of hu-BLT mice. Recombinant IL-2 (rhIL-2) was obtained from NIH-BRB. Flow cytometry and other antibodies used in the study were obtained from BioLegend (San Diego, CA). Human melanoma A375 cells were donated by Dr. Toru Hiraga (Department of Histology and Cell Biology, Matsumoto Dental University, Japan) [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] and were cultured in DMEM (Life Technologies) supplemented with 10% FBS and 2% antibiotic-antimycotic (Gemini Bio-Products). Oral squamous carcinoma stem cells (OSCSCs) were isolated from patients with tongue tumors at UCLA [\u003cspan additionalcitationids=\"CR62 CR63\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. OSCSCs were cultured in RPMI 1640 medium supplemented with 10% FBS, 2% Antibiotic/Antimycotic Solution (Cytiva, MA), 1.4% Sodium Pyruvate (Gibco, CA, USA), 1.4% MEM Non-Essential Amino Acids (Gibco, CA, USA), and 0.15% sodium bicarbonate. Antibodies that were used for flow cytometry \u0026ndash; IgG2, CD16, CD56, CD3, CD45, and MHC-class I were purchased from Biolegend (San Diego, CA, USA). Human NK cells, T cells, and monocyte purification kits were obtained from Stem Cell Technologies (Vancouver, BC, Canada). PKH26 staining kit was purchased from Sigma-Aldrich, St Louis, MO. Human ELISA kits for IFN-γ were purchased from Biolegend (San Diego, CA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePurification of human NK cells and monocytes\u003c/h3\u003e\n\u003cp\u003eWritten informed consents approved by the UCLA Institutional Review Board (IRB) were obtained, and all procedures were approved by the UCLA-IRB. NK cells and monocytes were negatively selected and isolated from PBMCs using the EasySep\u0026reg; Human NK cell enrichment kit and monocyte isolation kit, respectively, purchased from Stem Cell Technologies (Vancouver, BC, Canada). Isolated NK cells and monocytes were stained with anti-CD16 and anti-CD14 antibodies, respectively, to measure the cell purity using flow cytometric analysis.\u003c/p\u003e\n\u003ch3\u003eProbiotic bacteria AJ2 sonication\u003c/h3\u003e\n\u003cp\u003eGram-positive probiotic bacteria: AJ2 (Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei) were weighed and resuspended in RPMI 1640 containing 10% FBS at a concentration of 10 mg/1mL. The bacteria were thoroughly vortexed, then sonicated on ice for 15 seconds, set at a 60% amplitude. Sonicated samples were then incubated for 30 seconds on ice. After every five pulses, a sample was taken to observe under the microscope until at least 80 percent of the cell walls were lysed. It was determined that approximately 20 rounds of sonication/incubation on ice were conducted to achieve complete sonication. Finally, the sonicated sAJ2 samples were aliquoted and stored in a -80\u003csup\u003e○\u003c/sup\u003eC freezer.\u003c/p\u003e\n\u003ch3\u003eGeneration of osteoclasts and expansion of human NK cells\u003c/h3\u003e\n\u003cp\u003ePurified monocytes from human peripheral blood were cultured in alpha-MEM medium containing M-CSF (25 ng/mL) and RANKL (25 ng/mL) for 21 days, or otherwise specified. The medium was refreshed every 3 days with fresh alpha-MEM containing M-CSF and RANKL. Human purified NK cells were activated with rh-IL-2 (1000 U/ml) and anti-CD16mAb (3 \u0026micro;g/ml) for 18\u0026ndash;20 hours before they were co-cultured with osteoclasts and sonicated AJ2 for NK cells expansion. The medium was refreshed every 3 days with RMPI containing rh-IL-2 (1500 U/ml).\u003c/p\u003e\n\u003ch3\u003eAnalysis of melanoma growth in humanized-BLT mice\u003c/h3\u003e\n\u003cp\u003e Animal research was performed under the written approval of the UCLA Animal Research Committee (ARC). Humanized-BLT (hu-BLT; human bone marrow/liver/thymus) mice were prepared as previously described [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. For melanoma i\u003cem\u003en vivo\u003c/em\u003e growth, A375 human melanoma cells (1 x 10\u003csup\u003e6\u003c/sup\u003e) were mixed 1:1 with HC Matrigel (Corning, Corning, NY) and injected subcutaneously into the right flank of hu-BLT mice as described previously [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Tumor growth was measured biweekly with a caliper. sNK cells therapeutic group received four weekly tail vein injections of 2 x 10\u003csup\u003e6\u003c/sup\u003e sNK cells. Mice were euthanized when signs of morbidity were evident. Melanoma tumor, bone marrow, spleen, and peripheral blood were harvested from mice at the end of the experiment or when the tumor size reached 2cm in diameter.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell dissociation and cell culture of tissues from hu-BLT mice\u003c/h2\u003e \u003cp\u003eThe melanoma tumor harvested from hu-BLT mice were immediately cut into 1 mm\u003csup\u003e3\u003c/sup\u003e pieces and placed into a digestion buffer containing 1 mg/ml collagenase IV, 10 U/ml DNAse I, and 1% bovine serum albumin (BSA) in DMEM media, and incubated for 20 minutes at 37\u003csup\u003e˚\u003c/sup\u003eC oven on a 150 rpm shaker. After digestion, the sample was filtered through a 40 mm cell strainer and centrifuged at 1500 rpm for 10 minutes at 4˚C. The pellet was resuspended in DMEM media, and cells were counted. To obtain single-cell suspensions from BM, femurs were cut from both ends and flushed from one end to the other using RPMI media, BM cells were filtered through a 40 \u0026micro;m cell strainer. To obtain single-cell suspensions from the spleen, the spleen was smashed until no big piece was left, and the sample was filtered through a 40 \u0026micro;m cell strainer and centrifuged at 1500 rpm for 5 minutes at 4˚C. The pellet was re-suspended in ACK buffer to remove the red blood cells for 2\u0026ndash;5 minutes, followed by re-suspension in RMPI media and centrifuged at 1500 rpm for 5 minutes at 4˚C. Peripheral blood mononuclear cells (PBMCs) were isolated using ficoll-hypaque centrifugation of heparinized blood specimens. The buffy coat containing PBMCs was harvested, washed, and resuspended in RPMI 1640 medium.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePurification of NK cells and CD3 + T cells from hu-BLT mice\u003c/h3\u003e\n\u003cp\u003eNK cells and CD3\u0026thinsp;+\u0026thinsp;T cells from hu-BLT mice splenocytes were isolated using the human CD56\u0026thinsp;+\u0026thinsp;selection kit and human CD3\u0026thinsp;+\u0026thinsp;T selection kit, respectively (Stem Cells Technologies, Canada).\u003c/p\u003e\n\u003ch3\u003eEnzyme-linked immunosorbent assays (ELISAs) and multiplex cytokine assay\u003c/h3\u003e\n\u003cp\u003eThe assay was conducted as described in the manufacturer\u0026rsquo;s protocol. The plates were read in a microplate reader, at 450nm to obtain absorbance values (Biolegend, ELISA manual). To analyze and obtain the cytokine and chemokine concentration, a standard curve was generated by either two or three-fold dilution of recombinant cytokines provided by the manufacturer.\u003c/p\u003e \u003cp\u003eMultiplex assay was conducted as described in the manufacturer\u0026rsquo;s protocol for each specified kit. Analysis was performed using a Luminex multiplex instrument (MAGPIX, Millipore, Billerica, MA), and data was analyzed using the proprietary software (xPONENT 4.2, Millipore, Billerica, MA).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSurface Staining assays\u003c/h2\u003e \u003cp\u003eStaining was performed by labeling the cells with antibodies as described previously [\u003cspan additionalcitationids=\"CR69\" citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Briefly, the cells were washed twice with ice-cold PBS/1% BSA. Predetermined optimal concentrations of specific human flow cytometric antibodies were added to 1 x 10\u003csup\u003e4\u003c/sup\u003e cells in 50 \u0026micro;l of cold-PBS/1%BSA and cells were incubated on ice for 30 min. Thereafter, cells were washed in cold PBS/1%BSA and brought to 500 \u0026micro;l with PBS/1%BSA. Flow cytometry analysis was performed using a Beckman Coulter Epics XL cytometer (Brea, CA), and results were analyzed in FlowJo vX software (Ashland, OR).\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cb\u003e51\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eCr release cytotoxicity assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe \u003csup\u003e51\u003c/sup\u003eCr release assay was performed as described previously [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. We used OSCSCs as target cells to assess NK cell-mediated cytotoxicity because these cells are the most susceptible cells to NK cell-mediated cytotoxicity [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Briefly, different numbers of effector cells were incubated with \u003csup\u003e51\u003c/sup\u003eCr\u0026ndash;labeled target cells. After a 4-hour incubation period the supernatants were harvested from each sample and counted for released radioactivity using the gamma counter. The percentage specific cytotoxicity was calculated using the following formula:\u003c/p\u003e\u003cp\u003e\u003cimg 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\" style=\"width: 446px; height: 92.1681px;\" width=\"446\" height=\"92.1681\"\u003e\u003c/p\u003e\u003cp\u003eLytic unit 30/10\u003csup\u003e6\u003c/sup\u003e is calculated by using the inverse of the number of effector cells needed to lyse 30% of the tumor target cells X100.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAn unpaired, two-tailed student t-test was performed for the statistical analysis. One-way ANOVA using Prism-10 software was used to compare different groups. (n) denotes the number of mice used for each condition in the experiment. The following symbols represent the levels of statistical significance within each analysis: ***(p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001), **(p-value 0.001\u0026ndash;0.01), *(p-value 0.01\u0026ndash;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eInfusion of super-charged NK cells inhibited melanoma tumor growth in hu-BLT mice\u003c/h2\u003e \u003cp\u003eHu-BLT mice were generated, and human melanoma tumor cells were implanted subcutaneously in mice as described in the Materials and Methods section. In vivo tumor growth was monitored weekly basis, and we observed a lower tumor growth rate in the mice infused with sNK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Tumor-bearing mice infused with sNK cells did not exhibit morbidity and were able to climb in the cage, whereas untreated tumor-bearing mice became morbid and had complications in climbing and running. The tumor inhibiting potential of sNK cells was also validated by the small tumor size in the therapeutic group, post-sacrifice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). When we dissociated the tumor, a higher number of tumor cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and increased ex vivo tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) were observed in the tumor resected from untreated mice compared to sNK cell-treated group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003esNK cells circulated through PBMCs, spleen, and bone marrow and were found within the tumor of hu-BLT mice, and were responsible for the induction of in vivo differentiation of the melanoma tumors\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn order to track sNK cells in mouse tissues, sNK cells were labeled with PKH dye before infusion. Post-sacrifice, we detected sNK cells in peripheral blood (17.2%), spleen (29.6%), bone marrow (4.64%), and tumor (2.12%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). When tumor-infiltrating human NK and T cells were assessed, increased percentages of NK and NKT cells were found to be infiltrating in tumors of tumor-bearing mice infused with sNK cells when compared to untreated tumor-bearing mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Tumors were cultured ex vivo for 7 days, increased percentages of human CD45\u0026thinsp;+\u0026thinsp;immune cells were seen in both untreated and IL-2 treated tumors from tumor-bearing mice with sNK infusion compared to untreated tumor-bearing mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). We also have examined the surface expression of differentiation antigens MHC-class I on tumors, and found higher MHC-class I surface expression on tumors obtained from sNK cells infused tumor-bearing mice as compared to untreated tumor-bearing mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003esNK cell infusion increased percentages of NK cells and restored IFN-γ secretion within the tissue compartments of melanoma tumor-bearing mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHigher NK cell percentages in PBMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and spleen (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) were seen in tumor-bearing hu-BLT mice infused with sNK cells. There was no change or a slight decrease in percentages of T cells in peripheral blood (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and spleen (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). A different profile of NK and T cell percentages was seen in the BM of tumor-bearing mice injected with sNK. There was a slight decrease in NK cell percentages, whereas a slight increase in T cell percentages was observed (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) in tumor-bearing mice injected with sNK cells. Increased secretion of IFN-γ was observed in peripheral blood-derived serum (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), PBMCs (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and S1A, and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), BM (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), and spleen (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, and Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) of tumor-bearing mice injected with sNK cells. Increased secretion of other factors, except IL-10, was seen in peripheral blood-derived serum (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). PBMCs expressed increased secretion of factors except VEGF (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Spleen from tumor-bearing mice injected with sNK demonstrated increased secretion of factors determined in this study (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \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\u003eInfusion of sNK cells restored secretion of IFN-γ and other factors in the peripheral blood-derived serum of melanoma tumor-bearing hu-BLT mice\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\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=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSerum\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIFN-γ\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTNF-α\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIL-10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIL-12\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIL-17A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eIL-1b\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIL-2\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\u003eTumor\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.5 +/- 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 +/- 1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.5 +/- 16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 +/- 1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6 +/- 0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2 +/- 1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5 +/- 2.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTumor\u0026thinsp;+\u0026thinsp;sNK\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSerum\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eGMCSF\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMIP-1b\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eITAC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eFractalkine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eMIP-3A\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTumor\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e291.5 +/- 168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.5 +/- 0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16 +/- 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e109 +- 12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4 +/- 1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTumor\u0026thinsp;+\u0026thinsp;sNK\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e328\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e320\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInfusion of sNK cells restored secretion of IFN-γ and other factors in the spleen of melanoma tumor-bearing hu-BLT mice\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePBMCs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIFN-γ\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTNF-α\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIL-6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIFN-a\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVEGF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGMCSF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIP-10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eEotaxin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eFractalkine\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\u003eTumor\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e0.9 +/- 0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c3\"\u003e \u003cp\u003e2.5 +/- 1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e5 +/- 3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c5\"\u003e \u003cp\u003e10 +/- 5.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e57 +/- 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c7\"\u003e \u003cp\u003e0.5 +/- 0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e3.2 +/- 0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c9\"\u003e \u003cp\u003e3.8 +/- 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e14 +/- 5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTumor\u0026thinsp;+\u0026thinsp;sNK\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e2 +/- 0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c3\"\u003e \u003cp\u003e4.1 +/- 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e7.7 +/- 2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c5\"\u003e \u003cp\u003e24 +/- 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e33.6 +/- 12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c7\"\u003e \u003cp\u003e3.1 +/- 1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e6 +/- 2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c9\"\u003e \u003cp\u003e4.3 +/- 0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e22 +/- 7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e\u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\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=\"\u0026minus;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpleen\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIFN-γ\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTNF-α\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIL-6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIL-10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGMCSF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMCP-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMIP-1a\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMIP-1b\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMDC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eIP-10\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\u003eTumor\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 +/- 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c3\"\u003e \u003cp\u003e3.7 +/- 1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e1.9 +/- 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c5\"\u003e \u003cp\u003e2.5 +/- 0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c6\"\u003e \u003cp\u003e6.15 +/- 2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c7\"\u003e \u003cp\u003e5.3 +/- 3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e202 +/- 165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e63 +/- 17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c10\"\u003e \u003cp\u003e211 +/- 91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c11\"\u003e \u003cp\u003e14.7 +/- 4.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTumor\u0026thinsp;+\u0026thinsp;sNK\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.5 +/- 2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c3\"\u003e \u003cp\u003e6.9 +/- 1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e4.9 +/- 1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c5\"\u003e \u003cp\u003e24 +/- 18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c6\"\u003e \u003cp\u003e12.2 +/- 0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c7\"\u003e \u003cp\u003e14.1 +/- 2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e805 +/- 296\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e118 +/- 48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c10\"\u003e \u003cp\u003e405 +/- 147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c11\"\u003e \u003cp\u003e34 +- 21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIncreased NK cell-mediated cytotoxicity was observed in PBMCs (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and S1B) and BM-derived immune cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) from tumor-bearing mice infused with sNK cells. Different NK cell-mediated cytotoxicity profile was seen in the spleen; sNK cells infused groups showed decreased levels of NK cell-mediated cytotoxicity compared to the untreated tumor-bearing group (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). sNK cells-infused group also expressed an increased level of IFN-γ was observed in spleen-derived NK (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) and T cells (Fig. S2), and also expressed increased NK cell-mediated activity in spleen-derived NK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Overall, restored immune function was observed with sNK cells therapy in melanoma tumor-bearing mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe have previously shown that a single infusion of supercharged NK cells was able to significantly decrease or eliminate oral, pancreatic, and uterine cancers in hu-BLT mice [\u003cspan additionalcitationids=\"CR73 CR74\" citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. In this report, we extended those studies to melanoma tumors to see if there were any differences in response to sNK cells. In addition, we studied the effect of sNK cell therapy, not only within the tumor microenvironment, but also the effect on PBMCs, bone marrow, and spleen. Infusion of sNK cells resulted in increased numbers of circulating PKH-stained sNK cells in PBMCs, spleen, bone marrow, and the tumor (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Interestingly, the levels of sNK cells were the highest in the spleen, then PBMCs, and the least in bone marrow. There were increased levels of sNK cells in the tumor cells, although the percentages were lower in comparison to the other tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Infusion of sNK cells significantly decreased the size and the weight of the tumor and delayed the growth of the tumors (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). When single cells were prepared from the tumors and equal numbers of tumors from both groups were cultured for several days, much less tumor growth was seen in the tumor cells from tumor-implanted mice treated with sNK cells as compared to the untreated group. We then focused on the type of immune cells circulating in the tumor cells. After single-cell preparation of the tumors, the levels of CD3 and CD56 subpopulations were determined in the tumor microenvironment. There was an increased percentage of CD3-CD56\u0026thinsp;+\u0026thinsp;NK cells in addition to an increase in total CD3\u0026thinsp;+\u0026thinsp;T cells and CD3\u0026thinsp;+\u0026thinsp;CD56\u0026thinsp;+\u0026thinsp;NKT cells within the tumor cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). We then cultured the tumor cells with and without IL-2 treatment and measured the circulating CD45\u0026thinsp;+\u0026thinsp;immune cells. The proportion of CD45\u0026thinsp;+\u0026thinsp;immune cells was higher in sNK-infused tumor-implanted BLT mice as compared to tumor alone-implanted mice, and the addition of IL-2 further increased the levels of CD45\u0026thinsp;+\u0026thinsp;immune cells within the tumor cultures. We have previously shown that sNK-mediated increase in NK cells and subsequent induction of IFN-γ increase the levels of MHC-class I on the tumor cells. Accordingly, we observed higher expression of MHC-class I on tumors resected from tumors implanted in hu-BLT mice infused with sNK cells. Treatment of the tumors resected from tumor-implanted hu-BLT mice infused with sNK and treated with IL-2 substantially increased the levels of MHC-class I expression when compared to tumors alone implanted mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These studies indicated that sNK cells circulate within the tumor and are likely responsible in part for the increased MHC-class I levels on tumors, which we have shown to be part of the four major biomarkers indicating the levels of differentiation of the tumor cells [\u003cspan additionalcitationids=\"CR75\" citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe then assessed the levels of circulating NK cells in PBMCs and found that the levels of NK cells were elevated in tumor-implanted mice infused with sNK cells. Due to the higher variability between mice, we could not show statistical significance, even though on average the levels were much higher in tumor-implanted mice infused with sNK cells as compared to only tumor-implanted mice. This observation is very important since we see the same trend in human patients infused with sNK cells, in which case the percentages of NK cells rise significantly and remain high for greater than 1 year (manuscript submitted). Based on chip analysis in humans, the elevated NK cells are from the recipient (autologous) than the donor's sNK cells. In this study, the donor's sNK cells were from male donors, and the recipient was female; therefore, in the chip assay, we could only see the X chromosome and not the Y chromosome, indicating that the elevated percentages of NK cells were autologous and not allogeneic. We then assessed the levels of IFN-γ secretion and NK cell-mediated cytotoxicity by PBMCs and found that to be significantly higher levels of IFN-γ and cytotoxicity against NK-specific targets (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, and S1). Interestingly, no difference could be seen for the levels of T cells within PBMCs of mice implanted with a tumor and infused with sNK cells as compared to tumor alone implanted mice. In addition, when other cytokines and chemokines were assessed in supernatants recovered from PBMCs, we have not only observed higher IFN-γ and TNF-α release, but also many other cytokines and chemokines were upregulated in tumor mice infused with sNK cells as compared to tumor mice alone (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). We have shown previously that the combination of IFN-γ and TNF-α elevated MHC-class I, CD54, and PDL1, whereas it decreased CD44 expression, a profile which was used to distinguish between stem-like cells and their differentiated counterparts [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Differentiation of tumor cells with IFN-γ and TNF-α slowed tumor growth substantially [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to PBMCs, we also observed similar patterns of increased cytokine and chemokine release in the serum of tumor-implanted hu-BLT mice that received infusion of sNK cells as compared to tumor alone-implanted mice (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Both IFN-γ and TNF-α release were upregulated in tumor mice receiving sNK cells (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In contrast, the circulating NK cells in bone marrow were on average lower in tumor mice infused with sNK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). At the moment it is unclear why bone marrow exhibits differently from PBMC and spleen (please see below). However, in accordance with PBMCs and spleen, the bone marrow cells exhibited significantly higher secretion of IFN-γ and mediated higher NK cell-mediated cytotoxicity from tumor mice infused with sNK cells as compared to tumor alone mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). As for the spleen, it exhibited higher percentages of NK cells in tumor mice infused with sNK cells, and the cells mediated increased secretion of IFN-γ while mediating lower cytotoxicity as compared to tumor alone implanted mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Similarly, we saw higher secretion of cytokines and chemokines from splenocytes when assessed by multiplex assay (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe then sorted the NK cells and T cells from the spleens of the mice and performed functional assays. We observed significantly increased secretion of INF-γ and augmented cytotoxicity by the NK cells sorted from tumor mice infused with the sNK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Likewise, sorted T cells were also able to increase the levels of IFN-γ from tumor mice infused with sNK cells, although due to high variability between mice, we could not achieve statistical significance (Fig. S2). We have shown previously that sNK cells preferentially expand CD8\u0026thinsp;+\u0026thinsp;T cells and increase their functional capabilities [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOverall, these results suggested that sNK cell therapy should be efficacious in melanoma and should be considered as a cell therapy to stop or eliminate these tumors. In addition, sNK cells, by increasing the levels and function of autologous NK cells, should be able to restore the inactivated NK cells by the tumor cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, and Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e)[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan additionalcitationids=\"CR73\" citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e].\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eNK cells: Natural killer cells\u003c/p\u003e\n\u003cp\u003esNK cells: Supercharged NK cells\u003c/p\u003e\n\u003cp\u003eCSCs: Cancer-stem-like-cells\u003c/p\u003e\n\u003cp\u003eHu-BLT: Humanized-bone marrow/liver/thymus\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIFN-\u0026gamma;\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eInterferon-gamma\u003c/p\u003e\n\u003cp\u003eIL-2: Interleukin 2\u003c/p\u003e\n\u003cp\u003eOCs: Osteoclasts\u003c/p\u003e\n\u003cp\u003ePBMCs: Peripheral blood-derived mononuclear cells\u003c/p\u003e\n\u003cp\u003eOSCSCs: Oral squamous cancer stem-like cells\u003c/p\u003e\n\u003cp\u003eELISAs: Enzyme-Linked Immunosorbent Assays\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCr: Chromium \u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e: Protocols allowing the collection and use of human specimens for this study were approved by the UCLA Office of the Human Research Protection Program (IRB# 20-1659). Written informed consents, approved by the UCLA Institutional Review Board\u003cstrong\u003e\u0026nbsp;(\u003c/strong\u003eIRB#11-000781), were obtained from healthy individuals and ovarian cancer patients, and the UCLA-IRB approved all procedures. Animal research was performed under the written approval of the UCLA Animal Research Committee (ARC) in accordance with all federal, state, and local guidelines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: The authors provide consent for publication to be published in the Cancer Immunology, Immunotherapy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u0026nbsp;\u003c/strong\u003eThe data presented in the study is included in the main and supplementary files of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eKK generated and analyzed data, wrote, reviewed, and edited the manuscript. 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Jewett, \u003cem\u003eSupercharged NK Cell-Based Immuotherapy in Humanized Bone Marrow Liver and Thymus (Hu-BLT) Mice Model of Oral, Pancreatic, Glioblastoma, Hepatic, Melanoma and Ovarian Cancers.\u003c/em\u003e Crit Rev Immunol, 2023. \u003cstrong\u003e43\u003c/strong\u003e(2): p. 13-25.\u003c/li\u003e\n\u003cli\u003eKaur, K., et al., \u003cem\u003eSequential therapy with supercharged NK cells with either chemotherapy drug cisplatin or anti-PD-1 antibody decreases the tumor size and significantly enhances the NK function in Hu-BLT mice.\u003c/em\u003e Front Immunol, 2023. \u003cstrong\u003e14\u003c/strong\u003e: p. 1132807.\u003c/li\u003e\n\u003cli\u003eKaur, K., et al., \u003cem\u003eProbiotic-Treated Super-Charged NK Cells Efficiently Clear Poorly Differentiated Pancreatic Tumors in Hu-BLT Mice.\u003c/em\u003e Cancers (Basel), 2019. \u003cstrong\u003e12\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eKaur, K., et al., \u003cem\u003eSuper-charged NK cells inhibit growth and progression of stem-like/poorly differentiated oral tumors in vivo in humanized BLT mice; effect on tumor differentiation and response to chemotherapeutic drugs.\u003c/em\u003e Oncoimmunology, 2018. \u003cstrong\u003e7\u003c/strong\u003e(5): p. e1426518.\u003c/li\u003e\n\u003cli\u003eBui, V.T., et al., \u003cem\u003eAugmented IFN-\u0026gamma; and TNF-\u0026alpha; Induced by Probiotic Bacteria in NK Cells Mediate Differentiation of Stem-Like Tumors Leading to Inhibition of Tumor Growth and Reduction in Inflammatory Cytokine Release; Regulation by IL-10.\u003c/em\u003e Front Immunol, 2015. \u003cstrong\u003e6\u003c/strong\u003e: p. 576.\u003c/li\u003e\n\u003cli\u003eKaur, K., A.P. Celis, and A. Jewett, \u003cem\u003eNatural Killer Cell-Secreted IFN-\u0026gamma; and TNF-\u0026alpha; Mediated Differentiation in Lung Stem-like Tumors, Leading to the Susceptibility of the Tumors to Chemotherapeutic Drugs.\u003c/em\u003e Cells, 2025. \u003cstrong\u003e14\u003c/strong\u003e(2): p. 90.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"NK cells, supercharged NK cells, cytotoxicity, melanoma, IFN-γ, humanized-BLT mice, cancer stem cells (CSC), immunotherapy","lastPublishedDoi":"10.21203/rs.3.rs-6792338/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6792338/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe have previously shown that a single infusion of supercharged NK cells (sNK) was able to significantly decrease or eliminate oral, pancreatic, and uterine cancers implanted in hu-BLT mice. In this report, we extended those studies to melanoma tumors to observe whether there were differences in response to sNK cells. Furthermore, we studied the effect of sNK cell therapy on PBMCs, bone marrow, and spleen of hu-BLT mice. Our studies indicated that infusion of super-charged NK cells inhibited melanoma tumor growth in hu-BLT mice. In addition, sNK cells circulated through PBMCs, spleen, and bone marrow, and they were also found within the tumor of hu-BLT mice. Moreover, the sNK cells were responsible for the induction of in vivo differentiation of the melanoma tumors. sNK cell infusion increased percentages of NK cells in PBMCs of hu-BLT mice, and restored cytotoxicity and IFN-γ secretion within the PBMCs, spleen, and bone marrow of melanoma tumor-bearing mice. Overall, these results suggested that sNK cell therapy is efficacious in limiting melanoma tumor growth and should be considered as a cell therapy to stop or eliminate these tumors in patients. In addition, sNK cells, by increasing the levels and function of autologous NK cells, should be able to restore the inactivated function of NK cells by the tumor cells in cancer patients.\u003c/p\u003e","manuscriptTitle":"Supercharged natural killer (NK) cells inhibit melanoma tumor progression and restore endogenous NK cell function in humanized BLT mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-09 08:31:54","doi":"10.21203/rs.3.rs-6792338/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":"723baf9e-209b-4ec9-91d5-bcbcc606e056","owner":[],"postedDate":"June 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-09T19:53:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-09 08:31:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6792338","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6792338","identity":"rs-6792338","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}