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Interferon-γ secreted by recruited Th1 cells in peritoneal cavity inhibits the formation of malignant ascites

Interferon-γ secreted by recruited Th1 cells in peritoneal cavity inhibits the formation of... www.nature.com/cddiscovery ARTICLE OPEN Interferon-γ secreted by recruited Th1 cells in peritoneal cavity inhibits the formation of malignant ascites 1,3 1,3 1,3 1 1 1 2 1 Chang Liu , Zhuanglong Xiao ,LiDu , Shenghua Zhu , Hongyu Xiang , Zehui Wang , Fang Liu and Yuhu Song © The Author(s) 2023 Type 1 T helper (Th1) cells generate an efficient antitumor immune response in multiple malignancies. The functions of Th1 cells in malignant ascites (MA) have not been elucidated. The distribution of helper T cells in peritoneal fluid and peripheral blood was determined in patients and animal models with malignant ascites. The effects of Th1-derived interferon-γ (IFN-γ) on the formation of malignant ascites were investigated. The mechanism underlying the recruitment of Th1 cells into peritoneal cavity was explored. In patients with malignant ascites and animal models of malignant ascites, the percentage of Th1 cells increased in peritoneal fluid compared with peripheral blood. Next, our experiment demonstrated that Th1 cells inhibited the growth of tumor cells by secreting −/− IFN-γ in vitro. In murine models of malignant ascites, increased peritoneal fluid and shorter survival time were observed in IFN-γ mice compared with wild-type (WT) mice. Then, the levels of C-X-C motif chemokine ligand (CXCL) 9/10 and the ratio of CXCR3 Th1 cells indicated the involvement of CXCL9, 10/CXCR3 axis in the recruitment of Th1 cells into peritoneal cavity. As expected, in −/− murine models of malignant ascites, the gradient between ascitic Th1 ratio and blood Th1 ratio decreased in CXCR3 mice compared with WT mice. IFN-γ secreted by recruited Th1 cells in peritoneal cavity inhibits the formation of malignant ascites. Hence, manipulation of Th1 cells or IFN-γ will provide a therapeutic candidate against malignant ascites. Cell Death Discovery (2023) 9:25 ; https://doi.org/10.1038/s41420-023-01312-5 BACKGROUND various cytokines and cellular interactions [11, 12]. Th1 cell has Malignant ascites account for about 5–25% of all cases of ascites been considered as an efficient CD4 T cell subset to generate and carry a poor prognosis. Malignant ascites is commonly antitumor immune response through different ways [9, 12]. It associated with a variety of neoplasms containing gastric, color- has been proven that Th1 cells participated in the formation of ectal, pancreatic, hepatobiliary, ovarian, endometrial, primary malignant pleural effusion [13–15]. Thus, we hypothesized peritoneal carcinomas [1–5]. Obviously, the formation of malig- Th1 cells participate in the pathogenesis of malignant ascites. nant ascites is a complex, multifactorial process. An imbalance However, the role of Th1 in malignant ascites was not reported between fluid secretion and absorption by peritoneum contri- yet. The function of Th1 cells in the formation of malignant butes to abnormal accumulation of fluid within peritoneal cavity. ascites were determined, and the mechanism underlying the Previous studies revealed malignant ascites results from the recruitment of Th1 cells into peritoneal cavity was explored in alteration in vascular permeability, the release of inflammatory our study. Our results demonstrated interferon γ produced by cytokines and the obstruction of lymphatic drainage [6, 7]. Th1 cell participated in the formation of malignant ascites However, the pathophysiology of malignant ascites has been through suppressing the growth of the tumor and reducing incompletely understood. Thus, further researches should be peritoneal permeability in vivo, and CXCL9,10/CXCR3 axis performed to explore the mechanism. mediated the recruitment of Th1 cells into peritoneal cavity The immune system can distinguish between normal cells through visceral peritoneum. and abnormal cells, and plays a critical role in fighting cancer. T lymphocytes, an essential part of immune system, are found in and around tumors, and seem to be critical in determining the RESULTS efficacy of immune surveillance [8, 9]. Helper T cells play a Significant increase of Th1 cells in malignant ascites central role in normal immune response by releasing factors Growing evidence has demonstrated helper T cells play that activate virtually all the other immune system cells [10, 11]. important roles in tumor immune surveillance [9, 12, 16]. Thus, Helper T cells are arguably important cells in adaptive the distribution of help T cells was initially determined in immunity. Recent studies demonstrated help T cell serves as a patients and animal models with malignant ascites. Firstly, key mediator through inducing efficient antitumor immune different subtypes of help T cells (Th1, Treg, Th2, Th17) in response [10, 12]. Helper T cells differentiate into Type 1 helper peripheral blood and peritoneal fluid were determined in T (Th1), Th2, Th17, and regulatory T (Treg) cells by exposure to patients with malignant ascites. Significant increases in 1 2 Division of Gastroenterology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. These authors contributed equally: Chang Liu, Zhuanglong Xiao, Li Du. email: yuhusong@163.com Received: 19 October 2022 Revised: 22 December 2022 Accepted: 9 January 2023 Official journal of CDDpress 1234567890();,: C. Liu et al. AB **** ascites blood Normal blood Patients with MA CD **** ascites blood Normal blood Rats with MA Fig. 1 Th1 cells in human and rat malignant ascites. A Representative dot plots from a patient with malignant ascites (MA) and a healthy + + control showing flow cytometric analysis of Th1 (IFN-γ CD4 ) cells; B the percentages of Th1 cells in peritoneal fluid, peripheral blood in patients with malignant ascites. The difference between two groups was determined by t test; C representative flow cytometric dot plots of Th1 cells from a rat with malignant ascites at 21 days after intraperitoneal injection of Walker-256 cells; D the percentages of Th1 cells in peritoneal fluid, peripheral blood in rat model of malignant ascites. The difference between two groups was analyzed by t test. ****p ≤ 0.0001. + + + + − Th1 cells (CD4 IFN-γ ), Treg cells (CD4 CD25 CD127 ), and significant increase of Th1 cells in peritoneal fluid compared + + Th17 cells (CD4 IL-17 ) were observed in malignant ascites with the corresponding blood in rat model of malignant ascites. compared with the corresponding blood (Fig. 1A, B and Fig. S1 Importantly, the proportions of different Th cell subtypes were and S2). Simultaneously, the results showed no significant analyzed in malignant ascites. In patients with malignant + + + differences in the percentage of Th2 cells (CD4 IL-4 ) between ascites, 32.7 % of CD4 lymphocyte was Th1 cell, 11.5% of + + peritoneal fluid and corresponding blood (Fig. S3). Secondly, CD4 lymphocyte was Treg cell, 1.8% of CD4 lymphocyte was the accumulation of peritoneal fluid and the tumors in Th2 cell; 2.6% of CD4 lymphocyte was Th17 cell (Fig. 1,Figs. peritoneal cavity were observed in rat model of malignant S1–3). Interestingly, similar ratio of Th1 cells in peritoneal fluid ascites (Fig. S4). In addition, the level of vascular endothelial was observed in rat model of malignant ascites (Fig. 1C, D). growth factor (VEGF) and angiotensin 2 (Ang-2), key cytokines These indicated Th1 cells accounted for the largest subtype of of peritoneal permeability, increased in peritoneal fluid helper T cells in malignant ascites. In addition, the gradient compared with peripheral blood (Fig. S5). All these confirmed between ascitic Th1 ratio and blood Th1 ratio was large. Given that rat model of malignant ascites was established success- these, Th1 cells were selected as the target subtype of helper fully. The results of flow cytometry (Fig. 1C, D) showed T cells in the pathogenesis of malignant ascites in our study. Cell Death Discovery (2023) 9:25 Normal control Rat with MA Normal control Patient with MA Blood Ascites Blood Blood Ascites Blood Th1/CD4+T cells Th1/CD4+T cells C. Liu et al. Th1 cells inhibits the growth of tumor cells used for murine determined in vitro. Naive CD4 T cells were purified from mouse model of malignant ascites by secreting IFN-γ in vitro spleen instead of malignant ascites since it is difficult to obtain Since the mice with a genetic disruption of Th1 cells is not sufficient number of naïve T cells from malignant ascites. Then, available, the effects of Th1 cells on the growth of tumor cells used naive T cells were differentiated into Th1 cells in the presence of for establishing murine model of malignant ascites were Th1 conditions [13]. Our results showed the purity of naïve CD4 Cell Death Discovery (2023) 9:25 C. Liu et al. Fig. 2 Th1 cells inhibit the growth of tumor cells for malignant ascites by secreting IFN-γ.A Flow cytometric analysis revealed that Th1 cells induced apoptosis of H22 cells and S180 cells in vitro after 48-h incubation with Th1 cells. Upper panel: representative flow cytometric dot plots of apoptosis assay (left) and the percentages of apoptotic cells (right) revealing Th1 cells induced apoptosis of H22 cells in vitro; lower panel: representative flow cytometric dot plots of apoptosis assay (left) and the percentages of apoptotic cells (right) revealing Th1 cells induced apoptosis of S180 cells in vitro. The difference between two groups was determined by t test. B proliferative activity assay showing Th1 cells suppressed the proliferation of H22 cells (upper) and S180 cells (lower). The difference between two groups was determined by t test. C Representative dot plots of flow cytometric analysis showing the percentage of CD4 cells in IFN-γ-producing cells in a patient with malignant ascites (left); the percentage of CD4 cells in IFN-γ-producing cells in patients with malignant ascites (right); D Representative dot plots of flow cytometric analysis showing the percentage of CD4 cells in IFN-γ-producing cells in a rat with malignant ascites (left); the percentage of CD4 cells in IFN-γ-producing cells in rats with malignant ascites (right). E Flow cytometric analysis revealed that IFN-γ induced apoptosis of H22 cells and S180 cells in vitro after 48-h incubation with IFN-γ. Upper panel: representative flow cytometric dot plots of apoptosis (left) and the percentage of apoptotic cells (right) revealing IFN-γ induced the apoptosis of H22 cells in vitro; lower panel: representative flow cytometric dot plots of apoptosis assay (left) and the percentage of apoptotic cells (right) revealing IFN-γ induced the apoptosis of S180 cells in vitro. The difference between two groups was determined by t test; F proliferative activity assay showing IFN-γ suppressed the proliferation of H22 cells (upper) and S180 cells (lower). The difference between two groups was determined by t test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. T cells was 92.6%, and the purity of Th1 cells reached 93.4% IFN-γ suppresses the growth of peritoneal carcinomatosis (Fig.S6). Subsequently, the tumor cells (H22 cells and S180 cells) in vivo used for murine models of malignant ascites were co-cultured In murine models of malignant ascites, peritoneal tumors were + −/− with naive CD4 T cells or Th1 cells for 48 h; and the apoptosis and found to reside on peritoneum in WT, IFN-γ mice administrated proliferative activity of tumor cells were evaluated. Apoptosis with H22 cells. The numbers of peritoneal tumor foci increased in −/− assay (Fig. 2A) showed that the proportion of apoptotic cells the IFN-γ mice group compared with the control (Fig. 4A, upper increased remarkably in tumor cells (H22 cells and S180 cells) panel). To evaluate the presence of peritoneal carcinomatosis in treated with Th1 cells compared with naïve CD4 T cells (p < 0.05). the living mice, PET-CT scan was performed in murine models of Proliferation assay (Fig. 2B) revealed that proliferative activity of malignant ascites. Increased radiotracer uptake on FDG-PET tumor cells was inhibited upon the treatment of Th1 cells. These scanning indicated tumors (Fig. 4B, upper panel). The PET imaging −/− results revealed that Th1 cells inhibited the growth of H22 and confirmed total lesion glycolysis increased in IFN-γ mice group S180 tumor cells in vitro. Interferon γ is a hallmark of Th1 compared with the control (Fig. 4C, upper panel). Interestingly, −/− lymphocytes, and IFN-γ promotes the development and function similar patterns were observed in WT, IFN-γ mice injected with of Th1 cells [12, 17–19]. Then, the percentage of CD4 cell in IFN- S180 cells (Fig. 4A–C, bottom panels). In addition, the survival of γ-producing cells was determined in ascites. Our results showed murine models was evaluated. The median survival times of WT, −/− 43.0% of IFN-γ-producing cells were Th1 cells in patients with IFN-γ mice bearing malignant ascites induced by the adminis- malignant ascites. In rat model of malignant ascites, 68.8% of IFN- tration of H22 cells were 18 and 14 days, respectively. Pairwise log γ-positive cells were Th1 cells (Fig. 2C, D). These indicated critical rank tests showed that there was significant difference in survival −/− role of Th1 in the production of IFN-γ in malignant ascites. Then, curve between IFN-γ group and WT mice administrated with the effects of IFN-γ on the growth of tumor cells used for murine H22 cells (Fig. 4D, left panel). Similar patterns were observed in −/− models of malignant ascites were investigated. As shown in Fig. WT, IFN-γ mice injected with S180 cells (Fig. 4D, right panel). All 2E, F, the proportion of apoptotic cells increased (Fig. 2E) and these showed that IFN-γ deficiency promoted the development of proliferative activity of tumor cells decreased (Fig. 2F) upon the peritoneal cancer and decreased the survival of mice with incubation of IFN-γ, indicating the tumor suppressive effect of IFN- malignant ascites. γ. These revealed that Th1 cells inhibited the growth of tumor cells by secreting IFN-γ. The recruitment of CXCR3 Th1 cells into peritoneal cavity via chemokine CXCL9 and CXCL10 Interferon γ inhibits the formation of malignant ascites in vivo Leukocyte recruitment is regulated by chemokines and their The above data have demonstrated Th1 suppressed the growth of corresponding chemokine receptor expressed in leukocyte tumor cells via IFN-γ in vitro, thus, the effect of IFN-γ on the [20, 21]. Therefore, we determined chemokines and chemokine formation of malignant ascites was determined in vivo. Murine receptors involved in the recruitment of Th1 cells into peritoneal models of malignant ascites were created by injecting of H22/ cavity. The samples of peripheral blood and ascites were collected −/− S180 cells into IFN-γ mice or the control. The accumulation of from patients, and then Th1-chemotaxin-related chemokines peritoneal fluid and peritoneal carcinomatosis were visible in IFN- (chemokine C-C motif ligand (CCL)3, CCL4, CCL5, CXCL9, CXCL10, −/− γ mice and the control (wild-type) (Figs. 3A, 4A). The weight of CXCL11) in peripheral blood and peritoneal fluid were determined −/− peritoneal fluid increased in IFN-γ mice compared with the (Fig. 5A). The results of ELISA showed the concentrations of the control mice (Fig. 3A). Then, peritoneal permeability was evaluated chemokines CXCL9 and CXCL10 were higher in malignant ascites since the increase of peritoneal permeability resulted in the than those in peripheral blood (Fig. 5A). Since CXCR3 is the formation of ascites. Peritoneal permeability was evaluated by the corresponding receptor for CXCL9 and CXCL10, CXCR3 expression leakage of Evan’s blue into peritoneal cavity. As shown in Fig. 3B, was determined in Th1 cells. Interestingly, the correlation analysis the concentration of Evan’s blue in ascites increased significantly showed the ratio of CXCR3 Th1 cells in peripheral blood −/− in IFN-γ mice compared with the control (wild-type). Finally, correlated positively with the gradient between ascitesTh1 ratio the levels of VEGF and ANG-2, key regulators of peritoneal and blood Th1 ratio (Fig. 5C, E, left panel). To further confirm our permeability, were determined. The results (Fig. 3C, D) showed the findings, chemokines and chemokine receptors in peripheral concentration of VEGF and ANG-II in peritoneal fluid increased in blood and ascites were determined in rat model of malignant −/− IFN-γ mice compared with the control. Therefore, murine ascites, similar results were found in rat model of malignant −/− models of malignant ascites using IFN-γ mice demonstrated ascites (Figs. 5B, D, E, right panel). The above results indicated that that IFN-γ inhibited the formation of malignant ascites. the CXCL9, 10/CXCR3 axis plays a key role in the migration of Cell Death Discovery (2023) 9:25 C. Liu et al. Fig. 3 Effects of IFN-γ on the formation of malignant ascites. A The weight of peritoneal fluid at 14 days after intraperitoneal injection of H22 or S180 cells showed that deficiency of IFN-γ promoted the formation of peritoneal fluid in murine model of malignant ascites; B peritoneal permeability assay demonstrated IFN-γ deficiency increased peritoneal permeability through determining Evan’s blue; C the −/− concentration of VEGF in peritoneal fluid and corresponding blood of WT or IFN-γ mice with malignant ascites; left panel: H22; right panel: −/− S180; D the concentration of angiotensin-2 in peritoneal fluid and corresponding blood of WT or IFN-γ mice with malignant ascites; left panel: H22; right panel: S180. The difference between two groups was determined by t test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns no significant. Th1 cells into peritoneal cavity. To prove the key role of CXCR3, plays an important role in the recruitment of Th1 cells into CXCR3 deficient mice received the administration of tumor cells to peritoneal cavity through visceral peritoneum. However, the establish murine models of malignant ascites. As expected, in increase in the numbers of peritoneal tumor foci and the weight −/− murine models of malignant ascites, the gradient between ascites of peritoneal fluid was not found in CXCR3 mice compared −/− Th1 ratio and blood Th1 ratio was lower in CXCR3 mice with wild-type mice (Fig. S8 and S9). All these revealed the compared with wild-type mice (Fig. 6A–D); which indicated critical recruitment of Th1 cells into peritoneal cavity via CXCL9, 10/ role of CXCR3 in the migration of Th1 cells into peritoneal cavity. CXCR3 axis. Another important issue is how Th1 cells in peripheral blood streamed into malignancy-affected peritoneal cavity. In rat model of malignant ascites, immunohistochemical staining of CD4 and DISCUSSION IFN-γ showed Th1 cells were preferentially located in visceral Malignant ascites is a common manifestation in the late stage of peritoneum, not in parietal peritoneum. It indicated Th1 cell gastrointestinal tract cancers and ovarian cancer [5]. Previous recruited into peritoneal cavity through visceral peritoneum (Fig. studies demonstrated Th1 cells mediated anti-cancer immunity S7). Then, the results of immunochemical staining revealed that a through different ways [9, 22]. While, the role of Th1 cells in decrease in the number of Th1 cells was observed in visceral malignant ascites has not been explored until now. Thus, the −/− peritoneum of CXCR3 mice compared with wild-type mice (Fig. details of Th1 cells in malignant ascites were investigated in our 6E). While, no significant change in the number of Th1 cells was study. The accumulation of Th1 cells in peritoneal fluid was shown in parietal peritoneum. These findings suggest that CXCR3 observed in patients with malignant ascites and animal model of Cell Death Discovery (2023) 9:25 C. Liu et al. Fig. 4 Effects of IFN-γ on the growth of peritoneal carcinomatosis and the survival of mice. A Representative photograph of WT and IFN-γ −/− mice after intraperitoneal injection of H22 and S180 cells. Marked abdomen expansion and multiple tumor foci were observed at 14 days after the administration of tumor cells. Representative photography showed most of tumor cells were found in visceral peritoneum; −/− B positron emission tomography and computed tomography imaging of WT and IFN-γ mice with malignant ascites; C total lesion −/− glycolysis (TLG) on PET/CT of WT and IFN-γ mice with malignant ascites. The difference between two groups was determined by t test; −/− D survival curve of WT and IFN-γ mice with malignant ascites. The difference between two groups was determined by pairwise log-rank tests; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns no significant. Cell Death Discovery (2023) 9:25 C. Liu et al. ns ns ** AB ns ascites blood Normal ascites blood Normal ascites blood Normal ascites blood Normal blood blood blood blood Patients with MA Rats with MA Rats with MA Patients with MA ns *** ascites blood Normal ascites blood Normal ascites blood Normal ascites blood Normal blood blood Patients with MA blood blood Patients with MA Rats with MA Rats with MA **** ** **** **** ascites blood Normal ascites blood Normal ascites blood Normal ascites blood Normal blood blood blood Rats with MA blood Patients with MA Rats with MA Patients with MA CD4 - FITC IFN γ -APC CXCR3 - PE CD4 - FITC IFN γ -APC CXCR3 - PE Patients Rats n =7 n = 8 r =0.8067 r = 0.7272 p =0.0284 p = 0.0409 (ascites-blood)Th1 (%) (ascites-blood)Th1 (%) malignant ascites, and Th1 cells inhibited the growth of tumor peritoneal cavity. In conclusion, our results demonstrated inter- cells by secreting IFN-γ in vitro. Interferon γ participated in the feron γ produced by Th1 cell participated in the formation of formation of malignant ascites through suppressing the growth of malignant ascites through suppressing the growth of the tumor the tumor and reducing peritoneal permeability in vivo. Impor- and reducing peritoneal permeability in vivo, and CXCL9,10/ tantly, further study demonstrated CXCL9, CXCL10/CXCR3 axis CXCR3 axis mediated the recruitment of Th1 cells into peritoneal played an important role in the recruitment of Th1 cells into cavity through visceral peritoneum (Fig. 6F). Cell Death Discovery (2023) 9:25 Patient with MA Ascites Blood Blood CXCR3 Th1/Th1 (%) Rat with MA Ascites Blood Blood CXCR3 Th1/Th1 (%) CXCL9 pg/ml C. Liu et al. Fig. 5 The expression of chemokines and receptors for recruitment of Th1 cells in patients and rats. A Th1-chemotaxin-related chemokines (CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11) in peritoneal fluid were determined by ELISA in patients with malignant ascites (MA). The difference between two groups was determined by t test; B the concentrations of Th1-chemotaxin-related chemokines (CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11) in peritoneal fluid in rat models of malignant ascites. The difference between two groups was determined by t test.; C representative flow cytometric dot plots showing CXCR3 Th1 cells in malignant ascites of patients compared with peripheral blood; D representative flow cytometric dot plots showing CXCR3 Th1 cells in malignant ascites of rat model compared with peripheral blood; E statistical analysis showing positive correlation between the ratio of serum CXCR3 Th1 cells, and ascites-blood gradient of Th1 ratio in patients (left) and rat models (right). The correlations were analyzed by Pearson correlation. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns no significant. CD4 T lymphocytes within the tumor microenvironment (TME) ablation inhibited the migration of Th1 cell into peritoneal cavity play important roles in tumor immune surveillance [12, 16]. using CXCR3-deficient mice. All these indicated CXCL-9,10/CXCR3 Previous studies demonstrated that multiple subgroups of CD4 axis was required for Th1 cell trafficking from peripheral blood to T cells such as Treg cells, Th1 cells, Th17 cells, Th22 cells and Th9 peritoneal cavity in malignant ascites. Simultaneously, our study cells play important immune regulatory roles in the pathogenesis showed that knockdown of CXCR3 expression had no significant of malignant pleural effusion (MPE) [13, 23–28]. Since major effect on the formation of malignant ascites and the growth of subtypes of CD4 T cells contained Th1, Th2, Th17 and Treg cells, tumor in peritoneal cavity in vivo. CXCR3 is expressed on natural the percentages of Th1, Th2, Th17 and Treg cells in malignant killer (NK) cells, B cells, and microvascular endothelial cells [29]. It is ascites were determined in our study. Th1 cells increased well-known that NK cell and B cells are involved in tumorigenesis significantly in peritoneal fluid compared with peripheral blood. and tumor progression in many malignancies. CXCR3 plays a Th1 cells accounted for the highest proportion of CD4 T cells in crucial role in the chemotaxis of Th1 cells, NK cells and B cells, peritoneal fluid. All these indicated Th1 cells was involved in the which leads to complex and divergent effects in the pathogenesis development of malignant ascites. Th1 cell was found within the of tumor [30]. In addition, CXCR3 is a double-edged sword in tumor microenvironment. Previous studies showed high numbers tumor progression [31]. of tumor-infiltrating Th1 cells has been identified as a good In conclusion, our study illustrated that Th1 cells migrated from prognostic marker in many types of cancers [9]. Th1 cells enhance peripheral blood to peritoneal cavity through visceral peritoneum, the functions of cytotoxic T lymphocyte (CTL), and recruit natural and IFN- γ produced by Th1 cells in peritoneal fluid inhibited the killer (NK) cells and type I macrophages to tumor microenviron- development of malignant ascites. Hence, manipulation of ment, which generates antitumor immune surveillance [9]. Th1 cells or IFN-γ will provide a therapeutic candidate against However, Th1 cells display tumor-promoting roles in some other malignant ascites. types of cancers, such as chronic myelogenous leukemia and colorectal carcinoma [9]. The details of Th1 cells in development of malignant ascites have been not reported until now; herein, we MATERIALS AND METHODS aimed to evaluate the effects of Th1 cells on the pathogenesis of Detailed materials and methods were provided in the online malignant ascites. The accumulation of Th1 cells in peritoneal fluid supplement Animal models of malignant ascites. A rat model of malignant ascites was was observed in malignant ascites, and Th1 cells inhibited the established through intraperitoneal administration of Walker 256 cell, a rat growth of tumor cells in vitro. Interferon γ, a hallmark of Th1 breast carcinoma cell line (2 × 10 cells per rat) into Sprague-Dawley rats lymphocytes [12, 17–19], plays a key role in the development and [32, 33]. Murine models of malignant ascites were made through function of Th1 cells [17, 19]. Our in vitro experiment demon- intraperitoneal injection of H22 cell, a murine hepatic carcinoma cell line strated that Th1 cells inhibited the growth of tumor cells by 6 6 (1 × 10 cells per mouse) or S180 cells (1 × 10 cells per mouse), a murine releasing IFN-γ. Then, the mice with the disruption of IFN-γ sarcoma cancer cell line [34–36]. All animal studies were approved by the showed IFN-γ inhibited the formation of malignant ascites and institutional animal care and use committee of Tongji Medical College, reduced peritoneal permeability. In addition, IFN-γ prolonged Huazhong University of Science and Technology. survival time in mouse model of malignant ascites. All these demonstrated that IFN-γ secreted by recruited Th1 cells displayed Sample collection and processing. Samples of ascites and serum were obtained from patients with malignant ascites and animal models of antitumor properties in murine model of malignant ascites. Lin H malignant ascites. The samples were collected in heparin-treated tubes, et al. found that elevated Th1 cell numbers in MPE predominantly and then subjected to further analysis. Samples of enrolled patients were produce IFN-γ and IFN-γ promoted the formation of MPE and −/− obtained during initial paracentesis of the patients with malignant ascites mouse death in IFN-γ mice [13]. The difference between our (17 patients) and the controls (17 patients). In addition, the samples were results and Lin’s study attributed to different models of serous also collected from animal models of ascites when animal models were membrane effusion, different types of tumors and different stages successfully created. The samples were incubated with suitable antibodies, of serous membrane effusion. Obviously, IFN-γ is a crucial cytokine and then analyzed on BD LSRFortessa X-20. Data were analyzed using implicated in anti-tumor immunity. IFN-γ possesses pro-apoptotic FlowJo 10.5.3. effects on tumor cells, facilitate Th1-driven cytotoxic T-cell response, promotes myeloid cell activation and antigen presenta- The effect of Th1 cells and IFN-γ on the growth of tumor cells tion [17, 19]. In addition, IFN-γ exhibited pro-tumorigenic effects in vitro under certain circumstances through novel cellular and molecular Naïve CD4 T cells were isolated from a single-cell suspension, which inflammatory mechanisms [17, 19]. The anti- and pro-tumorigenic was prepared from a 6-week-old C57/BL6 mouse spleen using the naïve functions of IFN-γ seems to be dependent on the contexts of CD4 T Cell Isolation Kit. Cell separation was performed either manually tumor specificity, microenvironmental factors, and signaling with MACS Columns or automatically with the auto MACS Pro Separator. Then, naïve CD4 T cells were incubated with IL-2, IL-12, and anti-IL-4 to intensity [19]. differentiate into Th1 cells. Tumor cells (H22 or S180 cells) were co- Chemokines play a vital role in recruitment of leukocytes to cultured with Th1 cells using the trans-well culture system [37]. The tumor microenvironments. Th1 cell migrated into peritoneal cavity effects of Th1 cells on the growth of tumor cells were evaluated by cell in response to a chemokine gradient. Firstly, concentration proliferation assay and apoptosis assay [38]. In addition, the effects of gradients of CXCL-9 and CXCL-10 between ascites and peripheral IFN-γ on the growth of tumor cells were determined by cell proliferation blood were observed. Further study demonstrated that CXCR3 assay and apoptosis assay [38]. Cell Death Discovery (2023) 9:25 C. Liu et al. Peritoneum A C F CXCR3 Vessel CD4 - FITC IFNγ -APC CD4 - FITC IFNγ -APC CXCL 9 Peritoneal cavity CXCL 10 IFN-γ CD4 - FITC IFNγ -APC CD4 - FITC IFNγ -APC The growth of tumor cell Ang-2 VEGF Th1 cell Peritoneal permeability CD4 - FITC IFNγ -APC CD4 - FITC IFNγ -APC Ascites CD4 - FITC IFNγ -APC CD4 - FITC IFNγ -APC H22 S180 ** B D *** -/- -/- WT CXCR3 WT CXCR3 H22 S180 -/- -/- Normal control WT CXCR3 WT CXCR3 The effects of IFN-γ on the formation of malignant ascites and formation of malignant ascites were determined through the weight of peritoneal fluid, peritoneal permeability. The effects of IFN-γ on the growth the growth of tumor cells in vivo of tumor cells were evaluated by gross pathology, histology, survival and Tumor cells (H22 cells and S180 cells) were injected intraperitoneally into −/− positron emission tomography and computed tomography (PET-CT) IFN-γ mice or wild-type (WT) mice; then the effects of IFN-γ on the images. Cell Death Discovery (2023) 9:25 -/- CXCR3 H22 WT H22 Ascites Blood Ascites Blood Parietal peritoneum Visceral peritoneum IFN-γ CD4 IFN-γ CD4 -/- CXCR3 S180 WT S180 Ascites Blood Ascites Blood C. Liu et al. Fig. 6 CXCR3 plays a key role in the migration of Th1 cells into peritoneal cavity. A the representative flow cytometric dot plots of Th1 cells −/− in peritoneal fluid and peripheral blood from WT and CXCR3 mice at 14 days after intraperitoneal injection of H22 cell; B the ascites- −/− peripheral blood gradient of Th1 ratio from WT and CXCR3 mice at 14 days after intraperitoneal injection of H22 cell. The difference between two groups was determined by t test; C representative flow cytometric dot plots of Th1 cells in peritoneal fluid and peripheral blood −/− from WT and CXCR3 mice at 14 days after intraperitoneal injection of S180 cell; D the ascites-peripheral blood gradient of Th1 ratio from −/− WT and CXCR3 mice at 14 days after intraperitoneal injection of S180; the difference between two groups was determined by t test; −/− E immunohistochemical staining revealed that a decrease of Th1 cells (CD4 + IFN-γ+) in visceral peritoneum of CXCR3 mice compared with wild-type mice; F Graphical summary of present research: CXCL9,10/CXCR3 axis mediated the recruitment of Th1 cells into peritoneal cavity, interferon γ secreted by Th1 cells in peritoneal fluid suppressed the growth of tumor cells. In addition, interferon γ reduced peritoneal permeability via VEGF and Ang-2. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns no significant; scale bars = 50 μm. 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Committee of Tongji Medical School (ChiCTR‐BOC‐17011724 (www.chictr.org.cn)). 41. Wang Z, Liu F, Tu W, Chang Y, Yao J, Wu W, et al. Embryonic liver fodrin involved in hepatic stellate cell activation and formation of regenerative nodule in liver cirrhosis. J Cell Mol Med. 2012;16:118–28. ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41420-023-01312-5. ACKNOWLEDGEMENTS Correspondence and requests for materials should be addressed to Yuhu Song. We thank Prof. Minjun Ji (Nanjing Medical University, Nanjing, China) for providing interferon-gamma (IFN-γ) gene-knockout mice. Reprints and permission information is available at http://www.nature.com/ reprints AUTHOR CONTRIBUTIONS Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims YS: designed the research and got a grant; C Liu, ZX, LD, SZ, HX, ZW: collected sample in published maps and institutional affiliations. and analyzed sample; C Li, ZX, LD, SZ, HX: performed animal experiments; C Liu, ZX, LD: performed cell experiments; C Liu, ZX, LD, SZ, HX, ZW, FL, YS: analyzed the data; C Liu, ZX, LD, FL, YS: wrote the paper. All authors read and approved the final paper. 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Interferon-γ secreted by recruited Th1 cells in peritoneal cavity inhibits the formation of malignant ascites

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www.nature.com/cddiscovery ARTICLE OPEN Interferon-γ secreted by recruited Th1 cells in peritoneal cavity inhibits the formation of malignant ascites 1,3 1,3 1,3 1 1 1 2 1 Chang Liu , Zhuanglong Xiao ,LiDu , Shenghua Zhu , Hongyu Xiang , Zehui Wang , Fang Liu and Yuhu Song © The Author(s) 2023 Type 1 T helper (Th1) cells generate an efficient antitumor immune response in multiple malignancies. The functions of Th1 cells in malignant ascites (MA) have not been elucidated. The distribution of helper T cells in peritoneal fluid and peripheral blood was determined in patients and animal models with malignant ascites. The effects of Th1-derived interferon-γ (IFN-γ) on the formation of malignant ascites were investigated. The mechanism underlying the recruitment of Th1 cells into peritoneal cavity was explored. In patients with malignant ascites and animal models of malignant ascites, the percentage of Th1 cells increased in peritoneal fluid compared with peripheral blood. Next, our experiment demonstrated that Th1 cells inhibited the growth of tumor cells by secreting −/− IFN-γ in vitro. In murine models of malignant ascites, increased peritoneal fluid and shorter survival time were observed in IFN-γ mice compared with wild-type (WT) mice. Then, the levels of C-X-C motif chemokine ligand (CXCL) 9/10 and the ratio of CXCR3 Th1 cells indicated the involvement of CXCL9, 10/CXCR3 axis in the recruitment of Th1 cells into peritoneal cavity. As expected, in −/− murine models of malignant ascites, the gradient between ascitic Th1 ratio and blood Th1 ratio decreased in CXCR3 mice compared with WT mice. IFN-γ secreted by recruited Th1 cells in peritoneal cavity inhibits the formation of malignant ascites. Hence, manipulation of Th1 cells or IFN-γ will provide a therapeutic candidate against malignant ascites. Cell Death Discovery (2023) 9:25 ; https://doi.org/10.1038/s41420-023-01312-5 BACKGROUND various cytokines and cellular interactions [11, 12]. Th1 cell has Malignant ascites account for about 5–25% of all cases of ascites been considered as an efficient CD4 T cell subset to generate and carry a poor prognosis. Malignant ascites is commonly antitumor immune response through different ways [9, 12]. It associated with a variety of neoplasms containing gastric, color- has been proven that Th1 cells participated in the formation of ectal, pancreatic, hepatobiliary, ovarian, endometrial, primary malignant pleural effusion [13–15]. Thus, we hypothesized peritoneal carcinomas [1–5]. Obviously, the formation of malig- Th1 cells participate in the pathogenesis of malignant ascites. nant ascites is a complex, multifactorial process. An imbalance However, the role of Th1 in malignant ascites was not reported between fluid secretion and absorption by peritoneum contri- yet. The function of Th1 cells in the formation of malignant butes to abnormal accumulation of fluid within peritoneal cavity. ascites were determined, and the mechanism underlying the Previous studies revealed malignant ascites results from the recruitment of Th1 cells into peritoneal cavity was explored in alteration in vascular permeability, the release of inflammatory our study. Our results demonstrated interferon γ produced by cytokines and the obstruction of lymphatic drainage [6, 7]. Th1 cell participated in the formation of malignant ascites However, the pathophysiology of malignant ascites has been through suppressing the growth of the tumor and reducing incompletely understood. Thus, further researches should be peritoneal permeability in vivo, and CXCL9,10/CXCR3 axis performed to explore the mechanism. mediated the recruitment of Th1 cells into peritoneal cavity The immune system can distinguish between normal cells through visceral peritoneum. and abnormal cells, and plays a critical role in fighting cancer. T lymphocytes, an essential part of immune system, are found in and around tumors, and seem to be critical in determining the RESULTS efficacy of immune surveillance [8, 9]. Helper T cells play a Significant increase of Th1 cells in malignant ascites central role in normal immune response by releasing factors Growing evidence has demonstrated helper T cells play that activate virtually all the other immune system cells [10, 11]. important roles in tumor immune surveillance [9, 12, 16]. Thus, Helper T cells are arguably important cells in adaptive the distribution of help T cells was initially determined in immunity. Recent studies demonstrated help T cell serves as a patients and animal models with malignant ascites. Firstly, key mediator through inducing efficient antitumor immune different subtypes of help T cells (Th1, Treg, Th2, Th17) in response [10, 12]. Helper T cells differentiate into Type 1 helper peripheral blood and peritoneal fluid were determined in T (Th1), Th2, Th17, and regulatory T (Treg) cells by exposure to patients with malignant ascites. Significant increases in 1 2 Division of Gastroenterology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. These authors contributed equally: Chang Liu, Zhuanglong Xiao, Li Du. email: yuhusong@163.com Received: 19 October 2022 Revised: 22 December 2022 Accepted: 9 January 2023 Official journal of CDDpress 1234567890();,: C. Liu et al. AB **** ascites blood Normal blood Patients with MA CD **** ascites blood Normal blood Rats with MA Fig. 1 Th1 cells in human and rat malignant ascites. A Representative dot plots from a patient with malignant ascites (MA) and a healthy + + control showing flow cytometric analysis of Th1 (IFN-γ CD4 ) cells; B the percentages of Th1 cells in peritoneal fluid, peripheral blood in patients with malignant ascites. The difference between two groups was determined by t test; C representative flow cytometric dot plots of Th1 cells from a rat with malignant ascites at 21 days after intraperitoneal injection of Walker-256 cells; D the percentages of Th1 cells in peritoneal fluid, peripheral blood in rat model of malignant ascites. The difference between two groups was analyzed by t test. ****p ≤ 0.0001. + + + + − Th1 cells (CD4 IFN-γ ), Treg cells (CD4 CD25 CD127 ), and significant increase of Th1 cells in peritoneal fluid compared + + Th17 cells (CD4 IL-17 ) were observed in malignant ascites with the corresponding blood in rat model of malignant ascites. compared with the corresponding blood (Fig. 1A, B and Fig. S1 Importantly, the proportions of different Th cell subtypes were and S2). Simultaneously, the results showed no significant analyzed in malignant ascites. In patients with malignant + + + differences in the percentage of Th2 cells (CD4 IL-4 ) between ascites, 32.7 % of CD4 lymphocyte was Th1 cell, 11.5% of + + peritoneal fluid and corresponding blood (Fig. S3). Secondly, CD4 lymphocyte was Treg cell, 1.8% of CD4 lymphocyte was the accumulation of peritoneal fluid and the tumors in Th2 cell; 2.6% of CD4 lymphocyte was Th17 cell (Fig. 1,Figs. peritoneal cavity were observed in rat model of malignant S1–3). Interestingly, similar ratio of Th1 cells in peritoneal fluid ascites (Fig. S4). In addition, the level of vascular endothelial was observed in rat model of malignant ascites (Fig. 1C, D). growth factor (VEGF) and angiotensin 2 (Ang-2), key cytokines These indicated Th1 cells accounted for the largest subtype of of peritoneal permeability, increased in peritoneal fluid helper T cells in malignant ascites. In addition, the gradient compared with peripheral blood (Fig. S5). All these confirmed between ascitic Th1 ratio and blood Th1 ratio was large. Given that rat model of malignant ascites was established success- these, Th1 cells were selected as the target subtype of helper fully. The results of flow cytometry (Fig. 1C, D) showed T cells in the pathogenesis of malignant ascites in our study. Cell Death Discovery (2023) 9:25 Normal control Rat with MA Normal control Patient with MA Blood Ascites Blood Blood Ascites Blood Th1/CD4+T cells Th1/CD4+T cells C. Liu et al. Th1 cells inhibits the growth of tumor cells used for murine determined in vitro. Naive CD4 T cells were purified from mouse model of malignant ascites by secreting IFN-γ in vitro spleen instead of malignant ascites since it is difficult to obtain Since the mice with a genetic disruption of Th1 cells is not sufficient number of naïve T cells from malignant ascites. Then, available, the effects of Th1 cells on the growth of tumor cells used naive T cells were differentiated into Th1 cells in the presence of for establishing murine model of malignant ascites were Th1 conditions [13]. Our results showed the purity of naïve CD4 Cell Death Discovery (2023) 9:25 C. Liu et al. Fig. 2 Th1 cells inhibit the growth of tumor cells for malignant ascites by secreting IFN-γ.A Flow cytometric analysis revealed that Th1 cells induced apoptosis of H22 cells and S180 cells in vitro after 48-h incubation with Th1 cells. Upper panel: representative flow cytometric dot plots of apoptosis assay (left) and the percentages of apoptotic cells (right) revealing Th1 cells induced apoptosis of H22 cells in vitro; lower panel: representative flow cytometric dot plots of apoptosis assay (left) and the percentages of apoptotic cells (right) revealing Th1 cells induced apoptosis of S180 cells in vitro. The difference between two groups was determined by t test. B proliferative activity assay showing Th1 cells suppressed the proliferation of H22 cells (upper) and S180 cells (lower). The difference between two groups was determined by t test. C Representative dot plots of flow cytometric analysis showing the percentage of CD4 cells in IFN-γ-producing cells in a patient with malignant ascites (left); the percentage of CD4 cells in IFN-γ-producing cells in patients with malignant ascites (right); D Representative dot plots of flow cytometric analysis showing the percentage of CD4 cells in IFN-γ-producing cells in a rat with malignant ascites (left); the percentage of CD4 cells in IFN-γ-producing cells in rats with malignant ascites (right). E Flow cytometric analysis revealed that IFN-γ induced apoptosis of H22 cells and S180 cells in vitro after 48-h incubation with IFN-γ. Upper panel: representative flow cytometric dot plots of apoptosis (left) and the percentage of apoptotic cells (right) revealing IFN-γ induced the apoptosis of H22 cells in vitro; lower panel: representative flow cytometric dot plots of apoptosis assay (left) and the percentage of apoptotic cells (right) revealing IFN-γ induced the apoptosis of S180 cells in vitro. The difference between two groups was determined by t test; F proliferative activity assay showing IFN-γ suppressed the proliferation of H22 cells (upper) and S180 cells (lower). The difference between two groups was determined by t test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. T cells was 92.6%, and the purity of Th1 cells reached 93.4% IFN-γ suppresses the growth of peritoneal carcinomatosis (Fig.S6). Subsequently, the tumor cells (H22 cells and S180 cells) in vivo used for murine models of malignant ascites were co-cultured In murine models of malignant ascites, peritoneal tumors were + −/− with naive CD4 T cells or Th1 cells for 48 h; and the apoptosis and found to reside on peritoneum in WT, IFN-γ mice administrated proliferative activity of tumor cells were evaluated. Apoptosis with H22 cells. The numbers of peritoneal tumor foci increased in −/− assay (Fig. 2A) showed that the proportion of apoptotic cells the IFN-γ mice group compared with the control (Fig. 4A, upper increased remarkably in tumor cells (H22 cells and S180 cells) panel). To evaluate the presence of peritoneal carcinomatosis in treated with Th1 cells compared with naïve CD4 T cells (p < 0.05). the living mice, PET-CT scan was performed in murine models of Proliferation assay (Fig. 2B) revealed that proliferative activity of malignant ascites. Increased radiotracer uptake on FDG-PET tumor cells was inhibited upon the treatment of Th1 cells. These scanning indicated tumors (Fig. 4B, upper panel). The PET imaging −/− results revealed that Th1 cells inhibited the growth of H22 and confirmed total lesion glycolysis increased in IFN-γ mice group S180 tumor cells in vitro. Interferon γ is a hallmark of Th1 compared with the control (Fig. 4C, upper panel). Interestingly, −/− lymphocytes, and IFN-γ promotes the development and function similar patterns were observed in WT, IFN-γ mice injected with of Th1 cells [12, 17–19]. Then, the percentage of CD4 cell in IFN- S180 cells (Fig. 4A–C, bottom panels). In addition, the survival of γ-producing cells was determined in ascites. Our results showed murine models was evaluated. The median survival times of WT, −/− 43.0% of IFN-γ-producing cells were Th1 cells in patients with IFN-γ mice bearing malignant ascites induced by the adminis- malignant ascites. In rat model of malignant ascites, 68.8% of IFN- tration of H22 cells were 18 and 14 days, respectively. Pairwise log γ-positive cells were Th1 cells (Fig. 2C, D). These indicated critical rank tests showed that there was significant difference in survival −/− role of Th1 in the production of IFN-γ in malignant ascites. Then, curve between IFN-γ group and WT mice administrated with the effects of IFN-γ on the growth of tumor cells used for murine H22 cells (Fig. 4D, left panel). Similar patterns were observed in −/− models of malignant ascites were investigated. As shown in Fig. WT, IFN-γ mice injected with S180 cells (Fig. 4D, right panel). All 2E, F, the proportion of apoptotic cells increased (Fig. 2E) and these showed that IFN-γ deficiency promoted the development of proliferative activity of tumor cells decreased (Fig. 2F) upon the peritoneal cancer and decreased the survival of mice with incubation of IFN-γ, indicating the tumor suppressive effect of IFN- malignant ascites. γ. These revealed that Th1 cells inhibited the growth of tumor cells by secreting IFN-γ. The recruitment of CXCR3 Th1 cells into peritoneal cavity via chemokine CXCL9 and CXCL10 Interferon γ inhibits the formation of malignant ascites in vivo Leukocyte recruitment is regulated by chemokines and their The above data have demonstrated Th1 suppressed the growth of corresponding chemokine receptor expressed in leukocyte tumor cells via IFN-γ in vitro, thus, the effect of IFN-γ on the [20, 21]. Therefore, we determined chemokines and chemokine formation of malignant ascites was determined in vivo. Murine receptors involved in the recruitment of Th1 cells into peritoneal models of malignant ascites were created by injecting of H22/ cavity. The samples of peripheral blood and ascites were collected −/− S180 cells into IFN-γ mice or the control. The accumulation of from patients, and then Th1-chemotaxin-related chemokines peritoneal fluid and peritoneal carcinomatosis were visible in IFN- (chemokine C-C motif ligand (CCL)3, CCL4, CCL5, CXCL9, CXCL10, −/− γ mice and the control (wild-type) (Figs. 3A, 4A). The weight of CXCL11) in peripheral blood and peritoneal fluid were determined −/− peritoneal fluid increased in IFN-γ mice compared with the (Fig. 5A). The results of ELISA showed the concentrations of the control mice (Fig. 3A). Then, peritoneal permeability was evaluated chemokines CXCL9 and CXCL10 were higher in malignant ascites since the increase of peritoneal permeability resulted in the than those in peripheral blood (Fig. 5A). Since CXCR3 is the formation of ascites. Peritoneal permeability was evaluated by the corresponding receptor for CXCL9 and CXCL10, CXCR3 expression leakage of Evan’s blue into peritoneal cavity. As shown in Fig. 3B, was determined in Th1 cells. Interestingly, the correlation analysis the concentration of Evan’s blue in ascites increased significantly showed the ratio of CXCR3 Th1 cells in peripheral blood −/− in IFN-γ mice compared with the control (wild-type). Finally, correlated positively with the gradient between ascitesTh1 ratio the levels of VEGF and ANG-2, key regulators of peritoneal and blood Th1 ratio (Fig. 5C, E, left panel). To further confirm our permeability, were determined. The results (Fig. 3C, D) showed the findings, chemokines and chemokine receptors in peripheral concentration of VEGF and ANG-II in peritoneal fluid increased in blood and ascites were determined in rat model of malignant −/− IFN-γ mice compared with the control. Therefore, murine ascites, similar results were found in rat model of malignant −/− models of malignant ascites using IFN-γ mice demonstrated ascites (Figs. 5B, D, E, right panel). The above results indicated that that IFN-γ inhibited the formation of malignant ascites. the CXCL9, 10/CXCR3 axis plays a key role in the migration of Cell Death Discovery (2023) 9:25 C. Liu et al. Fig. 3 Effects of IFN-γ on the formation of malignant ascites. A The weight of peritoneal fluid at 14 days after intraperitoneal injection of H22 or S180 cells showed that deficiency of IFN-γ promoted the formation of peritoneal fluid in murine model of malignant ascites; B peritoneal permeability assay demonstrated IFN-γ deficiency increased peritoneal permeability through determining Evan’s blue; C the −/− concentration of VEGF in peritoneal fluid and corresponding blood of WT or IFN-γ mice with malignant ascites; left panel: H22; right panel: −/− S180; D the concentration of angiotensin-2 in peritoneal fluid and corresponding blood of WT or IFN-γ mice with malignant ascites; left panel: H22; right panel: S180. The difference between two groups was determined by t test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns no significant. Th1 cells into peritoneal cavity. To prove the key role of CXCR3, plays an important role in the recruitment of Th1 cells into CXCR3 deficient mice received the administration of tumor cells to peritoneal cavity through visceral peritoneum. However, the establish murine models of malignant ascites. As expected, in increase in the numbers of peritoneal tumor foci and the weight −/− murine models of malignant ascites, the gradient between ascites of peritoneal fluid was not found in CXCR3 mice compared −/− Th1 ratio and blood Th1 ratio was lower in CXCR3 mice with wild-type mice (Fig. S8 and S9). All these revealed the compared with wild-type mice (Fig. 6A–D); which indicated critical recruitment of Th1 cells into peritoneal cavity via CXCL9, 10/ role of CXCR3 in the migration of Th1 cells into peritoneal cavity. CXCR3 axis. Another important issue is how Th1 cells in peripheral blood streamed into malignancy-affected peritoneal cavity. In rat model of malignant ascites, immunohistochemical staining of CD4 and DISCUSSION IFN-γ showed Th1 cells were preferentially located in visceral Malignant ascites is a common manifestation in the late stage of peritoneum, not in parietal peritoneum. It indicated Th1 cell gastrointestinal tract cancers and ovarian cancer [5]. Previous recruited into peritoneal cavity through visceral peritoneum (Fig. studies demonstrated Th1 cells mediated anti-cancer immunity S7). Then, the results of immunochemical staining revealed that a through different ways [9, 22]. While, the role of Th1 cells in decrease in the number of Th1 cells was observed in visceral malignant ascites has not been explored until now. Thus, the −/− peritoneum of CXCR3 mice compared with wild-type mice (Fig. details of Th1 cells in malignant ascites were investigated in our 6E). While, no significant change in the number of Th1 cells was study. The accumulation of Th1 cells in peritoneal fluid was shown in parietal peritoneum. These findings suggest that CXCR3 observed in patients with malignant ascites and animal model of Cell Death Discovery (2023) 9:25 C. Liu et al. Fig. 4 Effects of IFN-γ on the growth of peritoneal carcinomatosis and the survival of mice. A Representative photograph of WT and IFN-γ −/− mice after intraperitoneal injection of H22 and S180 cells. Marked abdomen expansion and multiple tumor foci were observed at 14 days after the administration of tumor cells. Representative photography showed most of tumor cells were found in visceral peritoneum; −/− B positron emission tomography and computed tomography imaging of WT and IFN-γ mice with malignant ascites; C total lesion −/− glycolysis (TLG) on PET/CT of WT and IFN-γ mice with malignant ascites. The difference between two groups was determined by t test; −/− D survival curve of WT and IFN-γ mice with malignant ascites. The difference between two groups was determined by pairwise log-rank tests; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns no significant. Cell Death Discovery (2023) 9:25 C. Liu et al. ns ns ** AB ns ascites blood Normal ascites blood Normal ascites blood Normal ascites blood Normal blood blood blood blood Patients with MA Rats with MA Rats with MA Patients with MA ns *** ascites blood Normal ascites blood Normal ascites blood Normal ascites blood Normal blood blood Patients with MA blood blood Patients with MA Rats with MA Rats with MA **** ** **** **** ascites blood Normal ascites blood Normal ascites blood Normal ascites blood Normal blood blood blood Rats with MA blood Patients with MA Rats with MA Patients with MA CD4 - FITC IFN γ -APC CXCR3 - PE CD4 - FITC IFN γ -APC CXCR3 - PE Patients Rats n =7 n = 8 r =0.8067 r = 0.7272 p =0.0284 p = 0.0409 (ascites-blood)Th1 (%) (ascites-blood)Th1 (%) malignant ascites, and Th1 cells inhibited the growth of tumor peritoneal cavity. In conclusion, our results demonstrated inter- cells by secreting IFN-γ in vitro. Interferon γ participated in the feron γ produced by Th1 cell participated in the formation of formation of malignant ascites through suppressing the growth of malignant ascites through suppressing the growth of the tumor the tumor and reducing peritoneal permeability in vivo. Impor- and reducing peritoneal permeability in vivo, and CXCL9,10/ tantly, further study demonstrated CXCL9, CXCL10/CXCR3 axis CXCR3 axis mediated the recruitment of Th1 cells into peritoneal played an important role in the recruitment of Th1 cells into cavity through visceral peritoneum (Fig. 6F). Cell Death Discovery (2023) 9:25 Patient with MA Ascites Blood Blood CXCR3 Th1/Th1 (%) Rat with MA Ascites Blood Blood CXCR3 Th1/Th1 (%) CXCL9 pg/ml C. Liu et al. Fig. 5 The expression of chemokines and receptors for recruitment of Th1 cells in patients and rats. A Th1-chemotaxin-related chemokines (CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11) in peritoneal fluid were determined by ELISA in patients with malignant ascites (MA). The difference between two groups was determined by t test; B the concentrations of Th1-chemotaxin-related chemokines (CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11) in peritoneal fluid in rat models of malignant ascites. The difference between two groups was determined by t test.; C representative flow cytometric dot plots showing CXCR3 Th1 cells in malignant ascites of patients compared with peripheral blood; D representative flow cytometric dot plots showing CXCR3 Th1 cells in malignant ascites of rat model compared with peripheral blood; E statistical analysis showing positive correlation between the ratio of serum CXCR3 Th1 cells, and ascites-blood gradient of Th1 ratio in patients (left) and rat models (right). The correlations were analyzed by Pearson correlation. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns no significant. CD4 T lymphocytes within the tumor microenvironment (TME) ablation inhibited the migration of Th1 cell into peritoneal cavity play important roles in tumor immune surveillance [12, 16]. using CXCR3-deficient mice. All these indicated CXCL-9,10/CXCR3 Previous studies demonstrated that multiple subgroups of CD4 axis was required for Th1 cell trafficking from peripheral blood to T cells such as Treg cells, Th1 cells, Th17 cells, Th22 cells and Th9 peritoneal cavity in malignant ascites. Simultaneously, our study cells play important immune regulatory roles in the pathogenesis showed that knockdown of CXCR3 expression had no significant of malignant pleural effusion (MPE) [13, 23–28]. Since major effect on the formation of malignant ascites and the growth of subtypes of CD4 T cells contained Th1, Th2, Th17 and Treg cells, tumor in peritoneal cavity in vivo. CXCR3 is expressed on natural the percentages of Th1, Th2, Th17 and Treg cells in malignant killer (NK) cells, B cells, and microvascular endothelial cells [29]. It is ascites were determined in our study. Th1 cells increased well-known that NK cell and B cells are involved in tumorigenesis significantly in peritoneal fluid compared with peripheral blood. and tumor progression in many malignancies. CXCR3 plays a Th1 cells accounted for the highest proportion of CD4 T cells in crucial role in the chemotaxis of Th1 cells, NK cells and B cells, peritoneal fluid. All these indicated Th1 cells was involved in the which leads to complex and divergent effects in the pathogenesis development of malignant ascites. Th1 cell was found within the of tumor [30]. In addition, CXCR3 is a double-edged sword in tumor microenvironment. Previous studies showed high numbers tumor progression [31]. of tumor-infiltrating Th1 cells has been identified as a good In conclusion, our study illustrated that Th1 cells migrated from prognostic marker in many types of cancers [9]. Th1 cells enhance peripheral blood to peritoneal cavity through visceral peritoneum, the functions of cytotoxic T lymphocyte (CTL), and recruit natural and IFN- γ produced by Th1 cells in peritoneal fluid inhibited the killer (NK) cells and type I macrophages to tumor microenviron- development of malignant ascites. Hence, manipulation of ment, which generates antitumor immune surveillance [9]. Th1 cells or IFN-γ will provide a therapeutic candidate against However, Th1 cells display tumor-promoting roles in some other malignant ascites. types of cancers, such as chronic myelogenous leukemia and colorectal carcinoma [9]. The details of Th1 cells in development of malignant ascites have been not reported until now; herein, we MATERIALS AND METHODS aimed to evaluate the effects of Th1 cells on the pathogenesis of Detailed materials and methods were provided in the online malignant ascites. The accumulation of Th1 cells in peritoneal fluid supplement Animal models of malignant ascites. A rat model of malignant ascites was was observed in malignant ascites, and Th1 cells inhibited the established through intraperitoneal administration of Walker 256 cell, a rat growth of tumor cells in vitro. Interferon γ, a hallmark of Th1 breast carcinoma cell line (2 × 10 cells per rat) into Sprague-Dawley rats lymphocytes [12, 17–19], plays a key role in the development and [32, 33]. Murine models of malignant ascites were made through function of Th1 cells [17, 19]. Our in vitro experiment demon- intraperitoneal injection of H22 cell, a murine hepatic carcinoma cell line strated that Th1 cells inhibited the growth of tumor cells by 6 6 (1 × 10 cells per mouse) or S180 cells (1 × 10 cells per mouse), a murine releasing IFN-γ. Then, the mice with the disruption of IFN-γ sarcoma cancer cell line [34–36]. All animal studies were approved by the showed IFN-γ inhibited the formation of malignant ascites and institutional animal care and use committee of Tongji Medical College, reduced peritoneal permeability. In addition, IFN-γ prolonged Huazhong University of Science and Technology. survival time in mouse model of malignant ascites. All these demonstrated that IFN-γ secreted by recruited Th1 cells displayed Sample collection and processing. Samples of ascites and serum were obtained from patients with malignant ascites and animal models of antitumor properties in murine model of malignant ascites. Lin H malignant ascites. The samples were collected in heparin-treated tubes, et al. found that elevated Th1 cell numbers in MPE predominantly and then subjected to further analysis. Samples of enrolled patients were produce IFN-γ and IFN-γ promoted the formation of MPE and −/− obtained during initial paracentesis of the patients with malignant ascites mouse death in IFN-γ mice [13]. The difference between our (17 patients) and the controls (17 patients). In addition, the samples were results and Lin’s study attributed to different models of serous also collected from animal models of ascites when animal models were membrane effusion, different types of tumors and different stages successfully created. The samples were incubated with suitable antibodies, of serous membrane effusion. Obviously, IFN-γ is a crucial cytokine and then analyzed on BD LSRFortessa X-20. Data were analyzed using implicated in anti-tumor immunity. IFN-γ possesses pro-apoptotic FlowJo 10.5.3. effects on tumor cells, facilitate Th1-driven cytotoxic T-cell response, promotes myeloid cell activation and antigen presenta- The effect of Th1 cells and IFN-γ on the growth of tumor cells tion [17, 19]. In addition, IFN-γ exhibited pro-tumorigenic effects in vitro under certain circumstances through novel cellular and molecular Naïve CD4 T cells were isolated from a single-cell suspension, which inflammatory mechanisms [17, 19]. The anti- and pro-tumorigenic was prepared from a 6-week-old C57/BL6 mouse spleen using the naïve functions of IFN-γ seems to be dependent on the contexts of CD4 T Cell Isolation Kit. Cell separation was performed either manually tumor specificity, microenvironmental factors, and signaling with MACS Columns or automatically with the auto MACS Pro Separator. Then, naïve CD4 T cells were incubated with IL-2, IL-12, and anti-IL-4 to intensity [19]. differentiate into Th1 cells. Tumor cells (H22 or S180 cells) were co- Chemokines play a vital role in recruitment of leukocytes to cultured with Th1 cells using the trans-well culture system [37]. The tumor microenvironments. Th1 cell migrated into peritoneal cavity effects of Th1 cells on the growth of tumor cells were evaluated by cell in response to a chemokine gradient. Firstly, concentration proliferation assay and apoptosis assay [38]. In addition, the effects of gradients of CXCL-9 and CXCL-10 between ascites and peripheral IFN-γ on the growth of tumor cells were determined by cell proliferation blood were observed. Further study demonstrated that CXCR3 assay and apoptosis assay [38]. Cell Death Discovery (2023) 9:25 C. Liu et al. Peritoneum A C F CXCR3 Vessel CD4 - FITC IFNγ -APC CD4 - FITC IFNγ -APC CXCL 9 Peritoneal cavity CXCL 10 IFN-γ CD4 - FITC IFNγ -APC CD4 - FITC IFNγ -APC The growth of tumor cell Ang-2 VEGF Th1 cell Peritoneal permeability CD4 - FITC IFNγ -APC CD4 - FITC IFNγ -APC Ascites CD4 - FITC IFNγ -APC CD4 - FITC IFNγ -APC H22 S180 ** B D *** -/- -/- WT CXCR3 WT CXCR3 H22 S180 -/- -/- Normal control WT CXCR3 WT CXCR3 The effects of IFN-γ on the formation of malignant ascites and formation of malignant ascites were determined through the weight of peritoneal fluid, peritoneal permeability. The effects of IFN-γ on the growth the growth of tumor cells in vivo of tumor cells were evaluated by gross pathology, histology, survival and Tumor cells (H22 cells and S180 cells) were injected intraperitoneally into −/− positron emission tomography and computed tomography (PET-CT) IFN-γ mice or wild-type (WT) mice; then the effects of IFN-γ on the images. Cell Death Discovery (2023) 9:25 -/- CXCR3 H22 WT H22 Ascites Blood Ascites Blood Parietal peritoneum Visceral peritoneum IFN-γ CD4 IFN-γ CD4 -/- CXCR3 S180 WT S180 Ascites Blood Ascites Blood C. Liu et al. Fig. 6 CXCR3 plays a key role in the migration of Th1 cells into peritoneal cavity. A the representative flow cytometric dot plots of Th1 cells −/− in peritoneal fluid and peripheral blood from WT and CXCR3 mice at 14 days after intraperitoneal injection of H22 cell; B the ascites- −/− peripheral blood gradient of Th1 ratio from WT and CXCR3 mice at 14 days after intraperitoneal injection of H22 cell. The difference between two groups was determined by t test; C representative flow cytometric dot plots of Th1 cells in peritoneal fluid and peripheral blood −/− from WT and CXCR3 mice at 14 days after intraperitoneal injection of S180 cell; D the ascites-peripheral blood gradient of Th1 ratio from −/− WT and CXCR3 mice at 14 days after intraperitoneal injection of S180; the difference between two groups was determined by t test; −/− E immunohistochemical staining revealed that a decrease of Th1 cells (CD4 + IFN-γ+) in visceral peritoneum of CXCR3 mice compared with wild-type mice; F Graphical summary of present research: CXCL9,10/CXCR3 axis mediated the recruitment of Th1 cells into peritoneal cavity, interferon γ secreted by Th1 cells in peritoneal fluid suppressed the growth of tumor cells. In addition, interferon γ reduced peritoneal permeability via VEGF and Ang-2. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns no significant; scale bars = 50 μm. 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Committee of Tongji Medical School (ChiCTR‐BOC‐17011724 (www.chictr.org.cn)). 41. Wang Z, Liu F, Tu W, Chang Y, Yao J, Wu W, et al. Embryonic liver fodrin involved in hepatic stellate cell activation and formation of regenerative nodule in liver cirrhosis. J Cell Mol Med. 2012;16:118–28. ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41420-023-01312-5. ACKNOWLEDGEMENTS Correspondence and requests for materials should be addressed to Yuhu Song. We thank Prof. Minjun Ji (Nanjing Medical University, Nanjing, China) for providing interferon-gamma (IFN-γ) gene-knockout mice. Reprints and permission information is available at http://www.nature.com/ reprints AUTHOR CONTRIBUTIONS Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims YS: designed the research and got a grant; C Liu, ZX, LD, SZ, HX, ZW: collected sample in published maps and institutional affiliations. and analyzed sample; C Li, ZX, LD, SZ, HX: performed animal experiments; C Liu, ZX, LD: performed cell experiments; C Liu, ZX, LD, SZ, HX, ZW, FL, YS: analyzed the data; C Liu, ZX, LD, FL, YS: wrote the paper. All authors read and approved the final paper. 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