Abstract Background Natural killer (NK) cells are recognized for their ability to kill tumor cells for tumor control, but tumor cells often develop resistance to evade NK cell-mediated cytotoxicity. Identification of molecular mechanisms by which tumor cells evade from NK cell-mediated killing may offer novel therapeutic strategies for potentiating NK-based cancer immunotherapy. Methods An in vitro tumor-NK cell co-culture system was employed to identify the most significantly altered genes in tumor cells following NK cell interaction. The cell death rate of tumor cells by NK cell exposure was quantified using flow cytometry. EL4 and HCT116 tumor models in C57BL/6, BALB/c-nu, and NOD/SCID mice were used for evaluating tumor growth differences induced by Rac2 knockdown or knockout. The cellular and molecular impact of Rac2 knockdown or knockout on the sensitivity of tumor cells to NK cell-mediated cytotoxicity was assessed using quantitative PCR, immunofluorescence, and mutation analysis. Results By screening expression levels of the Ras homology (Rho) GTPase family genes in tumor cells after co-culture with NK cells, we identified RAC2 as a key regulator of tumor cell resistance to NK cell-mediated cytotoxicity among the Rho GTPase family members. Furthermore, knockout of RAC2 in human colorectal cancer cells leads to increased tumor susceptibility to NK cell-mediated cytotoxicity in a xenograft tumor model. Mechanistically, the absence of RAC2 enhances tumor cell sensitivity to NK cell-mediated killing by facilitating cell–cell contact. Conclusions These findings indicate that the inhibition of RAC2 in tumor cells substantially enhances their susceptibility to NK cell-mediated cytotoxicity, thereby providing a potential therapeutic target for optimizing NK cell therapy. Keywords: Natural killer - NK, Immunotherapy __________________________________________________________________ WHAT IS ALREADY KNOWN ON THIS TOPIC * Natural killer (NK) cells, as components of the innate immune system, possess the capability to directly target and eliminate malignant cells. Nonetheless, tumor cells can circumvent NK cell-mediated cytotoxicity by modifying the expression of NK cell-inhibitory and activating ligands, or adhesion molecules on their surface. Identifying surface molecules on tumor cells that have great regulatory potential may enhance the susceptibility of these cells to NK cell-mediated destruction. RAC2, a member of the Ras homology (Rho) GTPase family, is known to govern cytoskeletal dynamics. However, the role of RAC2 in modulating the tumor cell cytoskeleton and its subsequent influence on the sensitivity of tumor cells to NK cell-mediated lysis remains inadequately investigated. WHAT THIS STUDY ADDS * This study identifies RAC2 as a key regulator of tumor cell resistance to NK cell-mediated cytotoxicity. Our findings demonstrate that Rac2 knockout enhances the sensitivity of tumor cells to NK cell-induced lysis through direct cell–cell contact. RAC2 knockout significantly amplifies the actin cytoskeletal response of tumor cell on NK cell engagement. Specifically, the aspartic acid residue 148 of RAC2 is crucial for the formation of the immunological synapse (IS) and the susceptibility of tumor cells to NK cell-mediated killing. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY * This study elucidates that the impact of RAC2 on tumor cell susceptibility to NK cell killing is contingent on direct cell–cell contact. The findings offer novel insights into the mechanism regulating tumor cell sensitivity to NK cytotoxicity and propose RAC2 as a potential target for NK cell-based antitumor immunotherapy. Introduction Natural killer (NK) cells, which are principal constituents of type 1 innate lymphoid cells, serve as the primary line of defense in antitumor immunity.[57]^1 Unlike adaptive immune cells, NK cells do not require prior stimulation by antigenic peptides. They possess the ability to independently recognize, attack, and eliminate tumor cells primarily through direct cell–cell contact with tumor cells and secretion of lytic granules, cytokines, and tumor necrosis factor. These mechanisms collectively induce apoptosis or cell death of tumor cells.[58]^2 Building on the inherent antitumor characteristics of NK cells, numerous studies have attempted to use NK cells for antitumor therapy. The advancement of NK cell-based antitumor immunotherapy focuses on enhancing their cytotoxicity and persistence in vivo, as well as optimizing the cellular sources used for adoptive transfer.[59]^3 4 Although NK cell therapy holds significant potential, tumor cells persistently evolve immune evasion strategies to circumvent or resist recognition and destruction by NK cells, consequently constraining the therapeutic efficacy of NK cell interventions. For example, tumor cells may modify their expression of adhesion molecules, thereby obstructing the access and adherence of NK cells to tumor cells,[60]^5 or produce immunosuppressive factors within the tumor immune microenvironment to suppress NK cell activity.[61]^6 Thus, to enhance NK cell-based immunotherapy, it is imperative to identify and target the specific tumor evasion mechanisms from NK cell attack. The immunological synapse (IS) is established when immune cells such as NK cells or CD8^+ T cells engage in communication with target cells to facilitate immune recognition.[62]^7 The establishment of an IS represents a highly dynamic process of cell–cell interaction, characterized by a series of sequential events, including cytoskeletal remodeling and actin cytoskeleton activity.[63]^8 9 NK cells are capable of inducing the lysis of target cells through the formation of IS, followed by the release of cytolytic granules.[64]^10 Ras homology (Rho) GTPases are pivotal signaling proteins in initiating a diverse array of immune functions by interacting with a wide spectrum of effectors and kinases.[65]^11 Through these mechanisms, Rho GTPases orchestrate molecular networks to regulate signaling pathways involved in diverse cellular functions, such as cytoskeletal modification, intracellular trafficking, cell migration, and cell proliferation. Specifically, IS regulation by Rho GTPase is achieved by facilitating alterations in actin and myosin within the cytoskeleton at the cell cortex.[66]^12 13 Through an analysis of prior studies on NK-tumor interactions, we identified that the Rho GTPase family proteins possess the potential to resist NK cell-mediated cytotoxicity. Specifically, in addition to previously reported CDC42,[67]^14 we identified another Rho GTPase family member, RAC2, in regulating tumor sensitivity to NK killing. RAC2 is able to maintain the dynamics of cell cytoskeleton remodeling.[68]^15 Prior research has demonstrated a significant association between RAC2 and the onset and progression of various cancers,[69]^16 17 especially cancers of hematopoietic origins. It plays a supportive role in multiple stages of hematopoietic cell proliferation, differentiation, and apoptosis, among other processes.[70]^18 The deletion or mutation of RAC2 significantly disrupts actin cytoskeleton remodeling and intracellular signaling.[71]^19 Recent studies have identified mutations in RAC2 among clinical patients exhibiting diverse immunological manifestations, including granulocyte deficiency and combined immunodeficiency.[72]^20 21 However, the role of RAC2 in the regulation of tumor cell cytoskeleton and its impact on tumor sensitivity to NK cells remains unexplored. We screened GTPase genes whose expression was upregulated in tumor cells following co-culture with NK cells, and identified RAC2 as a pivotal mediator of tumor resistance to NK cell-mediated cytotoxicity. In various tumor models, knockdown or knockout (KO) of Rac2 markedly enhanced the susceptibility of tumor cells to NK cell-mediated killing. Furthermore, inhibition of RAC2 expression in tumor cells significantly augmented the antitumor efficacy of NK cells in vivo, as demonstrated in xenografted mouse models. Notably, the enhanced NK sensitivity of tumor cells with RAC2 inhibition was primarily attributed to enhanced interactions between tumor cells and NK cells. Methods Mice, cells, and reagents 6–8 weeks old female C57BL/6 mice, BALB/c-nude mice and NOD/SCID mice were purchased from Beijing Vital River Laboratory Animal Technology. The human colorectal cancer cell lines SW480, SW620, HCT116, DLD-1, LS174T, and HT-29, along with the human erythroleukemia K562 cell line and HEK293T cells, were purchased from the American Type Culture Collection. The EL4 cell line, derived from C57BL/6 mouse lymphoma, was generously provided by Professor Wei Yang at Southern Medical University, Guangzhou, China. The human cervical cancer cell line HeLa was kindly provided by Professor Jun Cui at Sun Yat-sen University, Guangzhou, China. The NK-92MI cells, a human natural killer cell line stably expressing interleukin-2 (IL-2), were generously provided by Professor Tong Xiang at Sun Yat-sen University Cancer Center, Guangzhou, China. All cell lines were routinely screened to confirm the absence of mycoplasma contamination and were maintained in either Dulbecco’s Modified Eagle Medium or Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen), both supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin-streptomycin (Gibco, Cat# 15070063), in an incubator set at 37℃ with a 5% CO[2] atmosphere. Specifically, the NK-92MI cells were cultured in serum-free medium GT-T551 H3 (Takara) under the same incubation condition. Isolation of mouse NK cells from murine splenocytes: splenocytes were harvested from the spleens of 6-8 weeks old female C57BL/6 mice. The spleens were mechanically dissociated and filtered through a 70 µm cell strainer. NK cells were subsequently isolated from the splenocytes using a commercially available mouse NK cell isolation kit (Invitrogen, Cat# 8804-6828-74). After isolation, the mouse NK cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin, and recombinant murine IL-2 (20 ng/mL) (PeproTech, Cat# 212–12). Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood samples obtained from healthy male volunteers. The isolation was performed using density gradient centrifugation with Ficoll-Paque PLUS (GE Healthcare, Cat# 17-1440-03) as the lymphocyte isolation medium. The isolated PBMCs were subsequently cultured in RPMI 1640 medium, supplemented with 10% FBS, 10 mg/mL gentamycin (Sigma-Aldrich, Cat# 1914), 1 mM sodium pyruvate (Gibco, Cat# 11360070), and 2 mM GlutaMax (Gibco, Cat# 35050061), and stimulated with anti-Human CD3 (Thermo Scientific, 16-0037-38) and anti-Human CD8 Antibody (Thermo Scientific, 16-0281-38), and then the supernatant was collected after 2 days. Primary human NK cells were isolated from PBMCs samples using CD56 microbeads (Miltenyi Biotec, 130-050-401). After isolation, the human NK cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. The antibodies used for Western blot analysis included Anti-RAC2 (Santa Cruz Biotechnology, sc-517424), Anti-Flag-HRP (Sigma-Aldrich, sc-F1804), and anti-β-ACTIN (Santa Cruz Biotechnology, sc-47778). The carboxyfluorescein succinimidyl ester (CFSE) Cell Division Tracker Kit (Cat# 423801) and the Zombie NIR Fixable Viability Kit (Cat# 423105) were purchased from BioLegend. For the in vivo experiments, the anti-NK1.1 neutralizing antibody was purchased from Bio X Cell (Cat# BE0036). Fluorescence-activated cell sorting All flow cytometry assays were conducted using the BD FACSAria III cytometer. Prior to tumor cell co-culture, NK-92MI cells were labeled with CFSE dye, and subsequently co-cultured with HeLa cells at different ratios specified. Following co-culture, the cells were washed with phosphate-buffered saline (PBS) and resuspended in 1 mL of flow cytometry buffer. At the end of the co-culture period, HeLa and NK-92MI cells were separately sorted based on CFSE positivity. CRISPR/Cas9 knockout and shRNA/siRNA knockdown Specific gene KO cells were constructed using Clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) technology. Small-guide RNA (sgRNA) sequences were meticulously designed using the Optimized CRISPR design tool (available at [73]http://chopchop.cbu.uib.no/). The guide sequences are listed in [74]online supplemental table 1. The sgRNA was cloned into the Lenti-CRISPR V.2 vector, which also harbors the Streptococcus pyogenes Cas9 nuclease gene. The sgRNA vector was co-transfected with the pspax2 and pMD2.G packaging plasmids. The supernatant was harvested 48 hours after transfection, followed by a 2-day selection process using puromycin. The efficiency of the KO was assessed through analyses at both the messenger RNA (mRNA) and protein levels of target genes. The knockdown of Rac2 and β2m in tumor cells was achieved using the gene-specific small-hairpin RNA (shRNA), with the sequences provided in [75]online supplemental table 2. In brief, lentiviral vectors encoding the specific shRNAs were co-transfected with the packaging plasmids pspax2 and pMD2.G into HEK293T cells. Supernatants were collected 48 hours after transfection to obtain shRNA-expressing virus, which was subsequently used to infect EL4 or HeLa cells, followed by puromycin selection. The expression of Rac2 via a lentiviral vector was achieved by amplifying the coding sequence (CDS) of mouse Rac2 ([76]NC_000081.7) using PCR from a DNA fragment. This sequence was subsequently inserted into the pCDH-3xFLAG-EF1-copGFP-T2A-Puro vector to construct a lentiviral vector expressing mouse Rac2. Mouse β2M ([77]NC_000068.8) complementary DNA was inserted into the pLVX-IRES-mCherry expression vector. Primers used for Rac2 truncated mutants and point mutation constructions are listed in [78]online supplemental table 3, and were designed to construct lentiviral vectors for expression in EL4-sgRac2 cells. These cells were then selected using puromycin. EL4 cells infected with an empty vector were used as control. For the purpose of small interfering RNA (siRNA) knockdown, HeLa cells in the exponential growth phase were seeded into 6-well plates and subjected to transient transfection using siRNA and the RNAiMAX Transfection Reagent (Invitrogen, 13778150), following the manufacturer’s protocol. The sequences of the siRNAs used are listed in [79]online supplemental table 4. RNA isolation and quantitative real-time PCR Total RNA was extracted using Trizol reagent (Invitrogen, 15596018) in accordance with the manufacturer’s protocol. Subsequent reverse transcription of RNA was conducted employing a specific primer set and a reverse transcriptase reagent kit, which included a genomic DNA eraser. Quantitative real-time PCR was performed using the 2×PolarSignal SYBR Green mix Taq (MIKX, MKG900-01) and analyzed with a Bio-Rad CFX96 thermal cycler. The sequences of the primers are listed in [80]online supplemental table 5. Flow cytometry-based NK cytotoxicity assay CFSE^+ NK-92MI cells were co-cultured with tumor cells at specified effector-to-target (E:T) ratios. In the primary human NK cell cytotoxicity assay, NK cells were pre-stained with CFSE dye. In the murine cell cytotoxicity assay, we noticed that CFSE pre-staining of primary mouse NK cells could potentially alter their activity. Thus, mouse tumor cells were pre-stained with CFSE dye instead of primary mouse NK cells. In instances where inhibitor treatment was administered, tumor cells were pretreated with the inhibitors before being seeded into the culture wells. A comprehensive list of the inhibitors used is included in [81]online supplemental table 6. Co-cultures were incubated at 37℃ for specified time points, after which the cell mixtures were harvested for staining with Zombie NIR Fixable Viability Dye. Subsequently, the stained cell populations were analyzed via flow cytometry. The cytotoxic activity of the NK cells was quantified by subtracting the background signal of untreated target cells from that of all other samples within the same experimental cohort. The experiment was repeated three times, each setting consisting of three biological replicates. Transwell culture The suitable configuration of transwell inserts and corresponding 12-well plates were chosen to facilitate the separation of the two distinct cell types. The lower chamber was seeded with 500 µL of tumor cells and the upper chamber with 200 µL of NK cells. Following incubation at 37°C, the tumor cells were collected from the lower compartment and tumor cell death was assessed using flow cytometry. Western blot Cells were lysed using a lysis buffer supplemented with the PhosSTOP phosphatase inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and 1 mM dithiothreitol (DTT). Whole cell proteins were extracted by centrifugation at 12,000×g for 8 min, with all procedures conducted on ice to maintain protein integrity. The extracted proteins were resolved via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto a polyvinylidene difluoride membrane. The membrane was blocked with 5% bovine serum albumin for 1 hour at room temperature to prevent non-specific binding. The membrane was incubated with the primary antibody overnight at 4°C, followed by three washes with PBS containing Tween 20, then incubated with the secondary antibody for 1 hour at room temperature. Finally, the membrane was washed and subjected to chemiluminescence analysis using an ECL detection kit (Thermo Scientific, 32106). Mouse experiments In the tumor xenograft experiments, EL4 cells or EL4-sgRac2 cells (2×10^6 cells per mouse) were inoculated subcutaneously into the mice. Tumor volume was recorded and calculated using the formula 0.5 × tumor length × (tumor width)^2, where the longer dimension was designated as the tumor length. The body weight was also recorded. In the in vivo NK cell depletion experiments, an anti-NK1.1 antibody was administered intraperitoneally at a dosage of 100 µg/mouse on days −1, 2, and 5 following tumor inoculation. In the mouse colorectal cancer (CRC) xenograft experiments, a xenograft model using the human HCT116 cell line was established in NOD/SCID mice. HCT116 or HCT116-sgRAC2 cells (2×10^6 cells per mouse) were subcutaneously inoculated into the mice on day 0, with 12 mice in each group. When the tumor volume reached 100 mm^3, one group received intravenous injection of PBS, while the other group was administered with NK-92MI cells (1×10^7 cells per mouse). Three mice in each group were used for tumor tissue isolation and analysis of NK cell infiltration 24 hours after NK cell treatment. For analysis of the tumor-infiltrated NK-92MI cells, mouse tumors were dissociated by gentleMACS (Miltenyi Biotec, 130-093-235) and filtered through 70 µm cell strainers to generate single cell suspensions, then stained with anti-Human CD45 (BioLegend, 368524), anti-Human CD56 (BD Biosciences, 563041). The rest of the mice were monitored for tumor volumes every 2 days using a digital caliper. Animals were sacrificed on weight loss of ≥20% body weight or tumor ulceration. TUNEL assay The paraffin-embedded sections of mouse tumor tissues were stained using the DeadEnd Fluorometric Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Servicebio, G1504-50T) following the manufacturer’s protocol. Nuclear staining was performed with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime, C1002, 100 nmol/L) for 5 min at room temperature under light-protected conditions, followed by a PBS wash. Subsequently, images were acquired using a fluorescence microscope. Immunofluorescence Immunofluorescence imaging was conducted using a ZEISS LSM880 confocal microscope. Cells were seeded in a confocal cell culture dish (SPL Life Sciences, 200350) and maintained overnight at 37°C. Subsequently, the cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min. Following fixation, the cells were permeabilized with 0.2% Triton X-100 for 10 min and subjected to three times PBS washing. F-actin was then stained with rhodamine-phalloidin reagent (Cytoskeleton, PHDR1, 100 nmol/L in PBS) for 1 hour. Nuclei were stained with DAPI. Subsequently, images were acquired using a confocal fluorescence microscope. To facilitate the capture of an immunological synapse, NK92-MI cells were co-cultured with tumor cells at an E:T ratio of 2:1 for 30 min in a conical tube at 37°C. Subsequently, the cells were gently placed onto slides pre-coated with 0.1% poly-L-lysine (Sigma-Aldrich, Cat#P7280). Following a 30-min fixation period in 4% paraformaldehyde, immunofluorescence imaging was conducted as previously described. Conjugates were characterized by direct cell–cell contact between an effector cell (NK-92MI cell) and a target cell (HeLa cell or HeLa-sgRAC2 cell). The accumulation of F-actin at the contact site of these conjugates was referred to as synaptic accumulation. Quantification and statistical analysis The statistical data from this study were derived from triplicate experiments and were presented as “mean±standard error”. One-way analysis of variance (ANOVA) was employed to analyze differences when a single variable was present among groups, while a two-way ANOVA was used for bivariate analyses between groups. Comparisons between two datasets were conducted using a two-tailed unpaired t-test. Statistical significance was determined at a threshold of p<0.05. Data analysis, statistical computations, and graphical representations were conducted using GraphPad Prism V.8.0. Results NK killing stress induces high expression of RAC2 in tumor cells We performed a comprehensive bioinformatics analysis of differently expressed genes and pathway enrichment in tumor cells on NK cell killing by using the screening data from previous studies,[82]^22 23 and found that Rho GTPase family-related signaling pathways in tumor cells were significantly upregulated in response to NK cell killing, indicating a potential killing resistance function of these family proteins ([83]figure 1A). To investigate whether tumor cells can resist the NK cell killing by upregulating Rho GTPase family genes, we set up an NK-tumor co-culture screening system by using HeLa cells (a human cervical cancer cell line) as the target cells, and NK-92MI cells (an IL-2-self-supplying human NK cell line) as the effector cells (schematic in [84]figure 1B).[85]^24 The NK-92MI cells were prelabeled with CFSE dye, and then co-cultured with Hela cells for indicated time points at different E:T ratios. The viable HeLa cells after co-culture were sorted for follow-up analysis. First, quantitative PCR (qPCR) analysis of mRNA from the sorted HeLa cells showed undetectable levels of NK-specific genes such as NCAM-1, KLRK1 and KLRC1, confirming the purity of tumor cells without NK contamination ([86]online supplemental figure 1A–C). We next selected 16 genes from five Rho GTPase subfamilies to follow-up ([87]figure 1C), and the qPCR results showed that multiple genes from the Rho GTPase family were induced after NK-92MI cells co-culture, among which CDC42 and RAC2 are the most strikingly upregulated genes ([88]figure 1D,E, [89]online supplemental figure 2A–E). CDC42 has been previously reported for its function in mediating tumor cell resistance to NK killing.[90]^14 To further determine which one of the RAC family genes may play the central role in the resistance to NK cytotoxicity, we knocked down RAC2, RAC1, RAC3, and RHOG genes as well as CDC42 gene in HeLa cells by gene-specific siRNAs ([91]online supplemental figure 3A–E), and then tested the impact of siRNA-mediated gene knockdown on NK-92MI cell-mediated killing of HeLa-siRNA cells. Consistent with previous studies, we observed increased susceptibility of tumor cells to NK cell killing after CDC42 knockdown ([92]figure 1F, [93]online supplemental figure 4A,C). More strikingly, knockdown of the RAC2 gene showed the highest rate of tumor cell death after NK killing in different co-culture durations and E:T ratios ([94]figure 1G, [95]online supplemental figure 4B,D). Taken together, these results suggested that RAC2 is a pivotal Rho GTPase regulating tumor sensitivity to NK killing. Figure 1. NK killing stress induces high expression of RAC2 in tumor cells. (A) Pathway enrichment analysis on RNA sequencing result from NK killing resistance genes. (B) A schematic diagram illustrating the flow cytometry sorting procedure employed for the identification of NK cell resistance genes within the Rho GTPase family. (C) A schematic diagram illustrating 16 genes from five subfamilies of the Rho GTPase family. (D–E) q-PCR was used to detect the expression levels of CDC42 (D) and RAC2 (E) in HeLa cells following co-culture with NK-92MI cells. The P-sup group refers to the supernatant from PBMCs that were stimulated with anti-human CD3/CD28 monoclonal antibodies for 2 days. HeLa cells cultured with this supernatant served as a positive control. The HeLa-ctrl group was used as a control to account for any stress induced by the flow cytometry sorting process; this group consisted of HeLa cells individually cultured under the same flow cytometry sorting conditions as the experimental group. (F–G) HeLa cells treated with RAC1, RAC2, RAC3, RHOG, and CDC42 gene-specific siRNAs were used to detect cell death rate by FACS following co-culture with NK-92MI cells at 6 hours. The results are representative of at least three independent experiments. Vertical bars indicate mean±SEM. Statistical significance between groups was assessed using the one-way analysis of variance with Bonferroni’s post-test, *p<0.05; **p<0.01; ***p<0.001. ctrl, control; E:T, effector-to-target; CFSE, Carboxyfluorescein succinimidyl ester; FACS, fluorescence-activated cell sorting; h, hours; NK, natural killer; PBMCs, peripheral blood mononuclear cells; q-PCR, quantitative PCR; Rho, Ras homology; RFU, relative fluorescence units; siRNA, small interfering RNA. [96]Figure 1 [97]Open in a new tab RAC2 expression in tumor cells promotes tumor resistance to NK cytotoxicity We next validate the role of RAC2 in regulating tumor cell susceptibility to NK cell-mediated killing. We knocked out or knocked down the RAC2 gene in HeLa cells via sgRNA-based CRISPR/Cas9 technology or shRNA ([98]figure 2A). Consistently, the results showed that the sensitivity of tumor cells to NK-92MI cell killing was increased in HeLa cells with RAC2 ablation ([99]figure 2B). Furthermore, HeLa-sgRAC2 cells were more sensitive to primary human NK cells killing ([100]figure 2C). In line with human cells, knock-out or knock-down of the Rac2 gene in EL4 mouse lymphoma cells ([101]figure 2D) also increased their sensitivity to primary mouse NK cell cytotoxicity ([102]figure 2E). Furthermore, tumor cells treated with EHT-1864, a pan-inhibitor of RAC proteins,[103]^25 also showed significantly increased sensitivity to NK killing ([104]figure 2F). Taken together, these results confirm that RAC2 promotes tumor resistance to NK cytotoxicity. Figure 2. RAC2 expression in tumor cells promotes tumor resistance to NK cytotoxicity. (A) Western blot and q-PCR were used to assess the efficiency of RAC2 knockout or knockdown in HeLa-sgRAC2 and HeLa-shRAC2 cell lines. (B) NK-92MI cells were co-cultured with Hela, Hela-sgRAC2, and Hela-shRAC2 cells, while the K562 co-culture group served as the positive control. The rate of tumor cell death following co-culture was assessed by FACS. (C) HeLa WT and HeLa-sgRAC2 cells, were co-cultured with primary human NK cells, and the rate of tumor cell death following co-culture was assessed by FACS. (D) Western blot and q-PCR were used to verify the knockout or knockdown efficiency of Rac2 in EL4-sgRac2 and EL4-shRac2 cell lines. (E) EL4, EL4-sgRac2, EL4-shRac2 cells were co-cultured with primary mouse NK cells, and the rate of tumor cell death after co-culture was assessed using FACS, with the EL4-shβ2m co-culture group serving as the positive control. (F) Western blot was used to verify the inhibition efficiency of RAC2 in EL4 cells following treatment with EHT-1864. Additionally, the cell death rate in tumor cells was evaluated using FACS after co-culture with primary mouse NK cells. The results are representative of at least three independent experiments. Vertical bars indicate mean±SEM. Statistical significance between groups was assessed using the one-way analysis of variance with Bonferroni’s post-test, *p<0.05; **p<0.01; ***p<0.001. CFSE, Carboxyfluorescein succinimidyl ester; E:T, effector-to-target; FACS, fluorescence-activated cell sorting; h, hours; NK, natural killer; q-PCR, quantitative PCR; WT, wild type. [105]Figure 2 [106]Open in a new tab NK cells mediate the growth inhibition of RAC2-deficient tumors To investigate whether RAC2 resists NK cytotoxicity in vivo, we subcutaneously inoculated equal numbers of wild type (WT) or Rac2-KO EL4 cells into immune-competent C57BL/6 mice. Tumor growth of the Rac2-KO group was significantly slower compared with that of the WT group ([107]figure 3A). By contrast, Rac2 -KO did not impair the proliferation of EL4 cells in vitro ([108]online supplemental figure 3F). When EL4 tumor cells were inoculated into T cell-deficient nude mice, Rac2-KO tumors still grew significantly slower than WT tumors ([109]figure 3B). Notably, tumor growth of the Rac2-KO group, but not the WT group, was significantly increased after NK cells were depleted by an NK1.1-specific neutralizing antibody in C57BL/6 mice ([110]figure 3C). Taken together, these results indicate that NK cells are responsible for tumor growth inhibition of Rac2-KO tumors in vivo. Figure 3. NK cells mediate the growth inhibition of RAC2-deficient tumors. (A–B) C57BL/6 (A) mice and BALB/c-nude (B) mice were injected subcutaneously with EL4 and EL4-sgRac2 cells, n=5 per group, and tumor growth of the mice was monitored every other day. (C) EL4, EL4-sgRac2 cells were subcutaneously inoculated on C57BL/6 mice with or without anti-NK1.1 mAb treatment (n=5 per group), and tumor growth of the mice was monitored every other day. Tumor growth curves were analyzed by two-way analysis of variance with Bonferroni’s post-test, ns, not significant; *p<0.05; **p<0.01; ***p<0.001. i.p., intraperitoneal injection; mAb, monoclonal antibody; ko, knockout; NK, natural killer; PBS, phosphate-buffered saline; wt, wild type. [111]Figure 3 [112]Open in a new tab RAC2 knockout in human colon cancer cells enhanced the efficacy of adoptive NK therapy Our previous data demonstrated that RAC2-KO in both human and mouse tumor cells increased their sensitivity to NK cell killing. To further expand the translational potential of our findings, we then tested an adoptive NK cell therapy model using human CRC cells for tumor inoculation followed by NK infusion for tumor therapy.[113]^26 We first tested RAC2 protein expression level in several human CRC cell lines including HT-29, LS174T, DLD-1, SW620, SW480 and HCT116 cells, and found relatively high level expression of RAC2 protein in SW620, SW480 and HCT116 cells ([114]online supplemental figure 5A). We then knocked out RAC2 on these cell lines and repeated the in vitro NK killing assays, respectively. Consistent with previous findings, RAC2-KO significantly increased human CRC cells sensitivity to NK-92MI cell-mediated cytotoxicity ([115]figure 4A–C). In contrast, RAC2-KO did not impair tumor cell viability and proliferation of HCT116 cells when cultured alone ([116]online supplemental figure 5B). We next set up a xenograft mouse CRC model by inoculating HCT116 cells on NOD/SCID mice, a severely immune-compromised mouse model deficient in T, B and NK cells. Following subcutaneous injection of RAC2-WT (sgScr) or RAC2-KO (sgRAC2) HCT116 cells, the tumor growth rates were comparable between the two groups. NK-92MI cells were then injected intravenously into tumor-bearing mice for NK therapy ([117]figure 4D). As expected, adoptive transfer of NK-92MI cells significantly reduced tumor growth of both groups. Notably, RAC2-KO tumors were much more sensitive than WT tumors to NK-92MI cell therapy in vivo ([118]figure 4E). The frequencies of tumor-infiltrated NK cells were comparable between WT and RAC2-KO tumors 24 hours after NK infusion ([119]figure 4F), but the RAC2-KO group exhibited significantly higher levels of cell death within tumor tissues, as determined by the TUNEL assay ([120]figure 4G). These data confirmed the pivotal role of RAC2 in mediating CRC resistance to NK cell-based immunotherapy. Figure 4. RAC2 knockout in human colon cancer cells enhanced the efficacy of adoptive NK therapy. (A–C) Western blot was used to verify the RAC2 knockout efficiency. While the rate of tumor cell death after co-culture with NK-92MI cells was determined by FACS in SW620 (A), HCT116 (B) and SW480 cells (C). (D) A schematic illustration depicting the in vivo xenograft mouse model of human HCT116 colon cancer cells used for the NK cell therapy with NK-92MI cells infusion. (E) HCT116-sgScr and sgRAC2 cells were s.c. inoculated into NOD/SCID mouse (n=12). When the tumor volume reached 100 mm^3, six mice from each group received an adoptive transfer of NK-92MI cells or PBS via the tail vein. Tumor growth of the mice was monitored every other day. (F) Tumors were isolated, and tumor-infiltrating NK-92MI (human-CD45^+ CD56^+) cells were analyzed by flow cytometry (n=3). (G) Cell death within the tumor tissues from RAC2-WT/KO tumor-bearing mice was analyzed using the TUNEL assay following treatment with NK-92MI cells (n=3). Scale bars, 50 µm. The results are representative of at least three independent experiments. Vertical bars indicate mean±SEM. Tumor growth curves were analyzed by two-way analysis of variance with Bonferroni’s post-test. Statistical significance between groups was assessed using the one-way analysis of variance with Bonferroni’s post-test, or two-tailed unpaired t-test, ns, not significant; *p<0.05; **p<0.01; ***p<0.001. CFSE, carboxyfluorescein succinimidyl ester; DAPI, 4′,6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; i.v., intravenous; NK, natural killer; PBS, phosphate-buffered saline; s.c., subcutaneous; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. [121]Figure 4 [122]Open in a new tab Rac2 ablation increases tumor sensitivity to NK cytotoxicity depending on cell–cell contact We next investigated the underlying mechanism by which RAC2 regulates tumor sensitivity to NK cytotoxicity. First, we tested the possible cell death mode induced by NK killing by using specific inhibitors for ferroptosis (Fer-1, DFO), apoptosis (Z-VAD-FMK, a pan inhibitor for caspases), or necroptosis (NSA, NEC-1). Among these inhibitors, only Z-VAD-FMK totally abolished NK cell coculture-induced tumor cell death despite RAC2 status, suggesting that apoptosis is the major mode of NK-induced tumor cell death ([123]online supplemental figure 6A,B). We next measured the expression levels of major histocompatibility complex-I (MHC-I) molecule on tumor cells, a ligand for NK cell inhibitory receptors to prevent NK activation. Conversely, NK cells are activated and attack target cell with MHC-I deficiency.[124]27,[125]29 We observed a significant decrease in the expression of MHC-I protein on the surface of EL4 cells or Hela cells with RAC2 deficiency ([126]online supplemental figure 7A,B), indicating MHC-I downregulation is the main reason for RAC2-KO induced NK sensitivity. However, knockdown of RAC2 expression on K562 cells, an MHC-I-deficient human leukemia cell line,[127]^23 still increased K562 cell sensitivity to NK-92MI-mediated killing, suggesting that RAC2 regulates tumor sensitivity to NK cytotoxicity independent of MHC-I expression ([128]online supplemental figure 7C). To further exclude the possibility that MHC-I downregulation is the main cue for RAC2-KO induced NK sensitivity, we knocked down the expression of β2M, an essential component of MHC-I complex, by using shRNA targeting β2M in EL4 cells, and then restored B2M expression in these EL4-shβ2M cells ([129]online supplemental figure 7D,E). After co-culture with mouse primary NK cells, we found that neither knockdown nor restoring expression of B2M affected Rac2-dependent resistance to NK cell-mediated killing ([130]online supplemental figure 7F,G). Besides MHC-I binding to inhibitory receptors on NK cells, tumor cells also upregulate anti-apoptotic molecules and downregulate ligands for death receptors on NK cells to evade NK attack.[131]^30 We then compared mRNA levels of death receptor ligand molecules including Fas, Tnfr1, Trailr2, Bcl-2, and anti-apoptotic molecules Bcl-xl and Mcl-1 in WT and Rac2-KO EL4 cells, and found no significant difference in the expression levels of these genes ([132]online supplemental figure 7H). On ligand-receptor binding-induced death signaling transduction, NK cells mainly execute the killing effect by contacting target cells and forming an IS to release granulocytes into target cells. We next investigated whether RAC2 regulates cell–cell contact and IS formation between tumor cells and NK cells. We set up a transwell assay to do the tumor-NK co-culture experiment in separate chambers without cell-cell contact. Differing from significantly increased tumor cell death by NK co-culture in direct contact, tumor cells co-cultured with NK cells by transwell separation showed no increased cell death compared to that of tumor cell culture alone ([133]figure 5A,B). Moreover, the IS was notably increased between NK and RAC2-KO tumor cells compared to that of NK and WT tumor cells. These results demonstrated that RAC2 deficiency enhanced the tumor-NK contact and IS formation to boost tumor sensitivity to NK cell cytotoxicity. Figure 5. RAC2 ablation increases tumor sensitivity to NK cytotoxicity depending on cell-cell contact. (A–B) EL4 and EL4-sgRac2 cells were co-cultured with primary mouse NK cells, HeLa and HeLa-sgRAC2 cells were co-cultured with NK-92MI cells, with (A) or without (B) transwell chamber method, and tumor cell death rate was determined by FACS. (C–D) Confocal microscopy was used to observe the cytoskeletal morphology and F-actin fluorescence intensity of EL4, EL4-sgRac2 and EL4-shRac2 cells (n=8) (C) and SW620-sgScr, SW620-sgRAC2-1 and SW620-sgRAC2-2 cells (n=10) (D). Scale bars, 5 µm. (E–F) Confocal microscopy was used to observe the Hela and Hela-sgRAC2 cells (spindle cells) co-cultured with NK-92MI cells (round cells), representative immunofluorescent images (E) and quantitative analysis (F) for F-actin fluorescence intensity in HeLa, HeLa-sgRAC2 cells (n=11). Scale bars, 5 µm. (G) The ratio of the F-actin fluorescence intensity at the effector–target conjugate interface to the total fluorescence intensity of the unconjugated membranes. The results are representative of at least three independent experiments. Vertical bars indicate mean±SEM. Statistical significance between groups was assessed using the two-tailed t-test, or one-way analysis of variance with Bonferroni’s post-test, ns, not significant; *p<0.05; **p<0.01; ***p<0.001. DAPI, 4′,6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; MFI, mean fluorescence intensity; NK, natural killer; WT, wild type. [134]Figure 5 [135]Open in a new tab Rho GTPases family genes play a central role in regulating cytoskeletal remodeling, which maintains cytoskeletal stability and normal cell motility through F-actin assembly.[136]^31 The cytotoxicity of NK cells on target cells also relies on cytoskeletal remodeling, which in turn forms IS to induce target cell death.[137]^32 Next, we tested whether knockdown of RAC2 expression affected the F-actin assembly in tumor cells. Phalloidin staining of F-actin showed that the F-actin assembly was significantly reduced in tumor cells with RAC2-KO ([138]figure 5C,D). A mature lytic synapse is formed primarily by cytoskeletal polarization.[139]^33 When NK-92MI cells (round shape) were co-cultured with spindle-shaped HeLa cells ([140]figure 5E), we observed a significant decrease in F-actin fluorescence signal in the RAC2-KO HeLa cells compared to WT cells ([141]figure 5F). On the contrary, in HeLa-sgRAC2 cells, the cortical region within the E:T cells conjugate on average showed stronger F-actin signal compared with that of WT cells ([142]figure 5G). In conclusion, these results suggested that tight contact with NK cells induces a more intense F-actin response in tumor cells with RAC2 deficiency. Aspartic acid 148 residue of Rac2 is critical for IS formation and NK killing sensitivity of tumor cells Recent studies reported that the carboxyl-terminal CAAX motif of most Rho GTPase family proteins is modified by geranylgeranylation to regulate cytoskeletal remodeling,[143]34,[144]36 while the aspartic acid residues (148 and 150) within the polybasic motif of RAC2 are important for its intracellular localization, actin polarization, and chemotaxis in neutrophils.[145]^37 To determine the role of geranylgeranylation and the polybasic motif of RAC2 in regulating tumor cell sensitivity to NK cell cytotoxicity, we re-expressed RAC2-WT, the geranylgeranylation-deficient RAC2 mutant (CAAX motif deleted, CAAX^−), the D148E mutant, or D150G mutant into EL4-sgRac2 cells, respectively ([146]figure 6A,B). The results showed that re-expression of RAC2-WT, RAC2-CAAX^-− or RAC2-D150G in EL4-sgRac2 cells all suppressed the increased tumor cell death mediated by NK killing, while the RAC2-D148E mutant failed to do so ([147]figure 6C). Consistently, re-expression of RAC2-WT recovered fluorescent intensity of F-actin in EL4-sgRac2 tumor cells, while RAC2-D148E did not ([148]figure 6D). These results identified D148 as a critical amino acid residue for RAC2 function in mediating tumor cell resistance to NK killing, and the D to E mutation impaired such function, likely due to subtle structural change. Figure 6. Aspartic acid 148 residue of RAC2 is critical for IS formation and NK killing sensitivity of tumor cells. (A) Schematic re-presentation of the Rac2 truncation and mutation construction, with the truncated or mutated sites in red. (B–D) Western blot showed the construct expression in EL4 cells, EL4-sgRac2 and EL4-sgRac2+RAC2 WT/CAAX^-/D148E/D150G cells (B), and tumor cells were co-cultured with primary mouse NK cells, and the tumor cell death rate was determined by FACS (C). Confocal microscopy was used to observe the cytoskeletal morphology and F-actin fluorescence intensity in EL4, EL4-sgRac2, EL4-sgRac2+RAC2 WT and EL4-sgRac2+RAC2-D148G (n=5) (D). Scale bars, 10 µm. (E–F) Kaplan-Meier analysis of RAC2 expression in relation to clinical prognosis in patients with solid tumors, specifically COAD (E), and hematological malignancies, such as AML (F), as derived from TCGA dataset. The p value was calculated using the log-rank test. The results are representative of at least three independent experiments. Vertical bars indicate mean±SEM. Statistical significance between groups was assessed using the one-way analysis of variance with Bonferroni’s post-test, ns, not significant; *p<0.05; **p<0.01; ***p<0.001. AML, acute myeloid leukemia; COAD, colon adenocarcinoma; CFSE, carboxyfluorescein succinimidyl ester; DAPI, 4′,6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; IS, immunological synapse; MFI, mean fluorescence intensity; NK, natural killer; TCGA, The Cancer Genome Atlas; WT, wild type. [149]Figure 6 [150]Open in a new tab Finally, we investigated the clinical relevance of RAC2 expression and patients with cancer prognosis using The Cancer Genome Atlas database. Correlation analysis showed that high RAC2 expression is associated with a poor clinical prognosis in patients with solid malignancies (represented by colon adenocarcinoma) and hematological malignancies (represented by acute myeloid leukemia) ([151]figure 6E,F). Taken together, these results demonstrated that RAC2 relies on the D148 residue to regulate cell cytoskeletal remodeling and tumor sensitivity to NK cytotoxicity, and high expression of RAC2 in human cancers is associated with poor prognosis. Discussion Our present findings identified RAC2 as a major GTPase family member that was upregulated in tumor cells by NK cell co-culture. RAC2 KO significantly increased the sensitivity of tumor cells to NK cell-mediated killing. In xenograft tumor models, infusion of NK cells significantly inhibited tumor growth of RAC2-KO compared to that of WT tumor cells. Mechanistically, RAC2 KO or knockdown increased tumor sensitivity to NK killing independent of the surface expression level of MHC-I molecules, nor the ligands of death receptors. Instead, it impaired the F-actin intensity and facilitated IS formation to increase the NK killing sensitivity, which can be partially reversed by re-expression of RAC2-WT, but not the RAC2 D148 mutant into RAC2-deficient cells. Based on the above findings, we propose that RAC2 can be considered as a new target for NK cell therapy, and inhibiting RAC2 in tumor cells may significantly enhance the therapeutic efficacy of NK cell-based cancer treatment. NK cells are a key component of the innate immune system and play an important role in controlling the development of malignancies. The efficacy of antitumor immunotherapy with NK cells depends on the toxic effects of NK cells.[152]^4 Previous studies have investigated the sensitivity of different types of cancer to NK cells and the corresponding factors that determine their sensitivity.[153]^22 The NK killing resistance genes were discovered in the glioblastoma multiforme (GBM) model and were verified in the head and neck squamous cell carcinoma (HNSCC) model. The resistant state of cancer cells to NK killing was changed to a sensitive state by gene editing, and the antitumor efficacy of NK cells from GBM and HNSCC was improved.[154]^5 Nonetheless, the finding is mainly derived from these two solid tumor models and still needs to be verified in other tumor types including hematological malignancies. In our study, after NK cells co-culture, the expression levels of the RAC2 and CDC42 genes in tumor cells were found to be greatly increased. It has been reported that deletion of the CDC42 gene in breast cancer cells can induce cytoskeletal remodeling and convert NK-killing resistant cell lines to a sensitive state.[155]^14 In this study, RAC2 has a similar function to the CDC42 gene in regulating cytoskeleton remodeling but exhibits a stronger effect than CDC42 in regulating tumor cell sensitivity to NK killing. Tumor cells are able to respond to NK cell attack through the rapid accumulation of actin by NK cells near IS formed by cellular contact, a process called the “actin response”.[156]^38 39 Actin remodeling of tumor cells plays a critical role in the resistance of tumor cells to NK cell-mediated killing.[157]^40 As a member of the Rho GTPase family, RAC2 promotes cellular action by forming lamellipodia and folding the plasma membrane.[158]^41 RAC2 inhibition does not increase tumor sensitivity to NK killing through the common MHC-I or cell death pathways, but by altering the cytoskeletal morphology and weakening the F-actin strength of cancer cells. We observed a significant increase in the ratio of F-actin fluorescence intensity at the E:T conjugated interface to the sum of unconjugated membranes after RAC2-KO. These results suggest that RAC2-KO regulates IS formation by enhancing tumor cell “actin response”, thereby converting tumor cells from an NK-killing-resistant to a sensitive state. But how did RAC2 affect the strength of cellular F-actin? Based on previous work, we found that the C-terminal prenylation sites and polybasic motif on RAC2 protein have an important impact on RAC2 function.[159]^34 Mutation of D148 in the RAC2 polybasic motif induced a significant change in the morphology of the tumor cytoskeleton and significantly increased the sensitivity of tumor cells to NK killing, highlighting the critical role of this residue in RAC2-mediated tumor sensitivity to NK killing. In conclusion, we identified RAC2 as an NK resistance gene of tumor cells. RAC2 inhibition in tumor cells increased the sensitivity of tumor cells to both in vitro NK cell killing and NK-based cell therapy in vivo. Mutation of D148 within the polybasic motif of RAC2 in tumor cells increased tumor sensitivity to NK cell killing. Therefore, we propose that RAC2 is a new target for NK cell-based cancer therapy by increasing the sensitivity of tumor cells to NK killing. Supplementary material online supplemental file 1 [160]jitc-13-5-s001.doc^ (3.1MB, doc) DOI: 10.1136/jitc-2024-010931 Acknowledgements