Progress on immuno-microenvironment and immune-related therapies in patients with pseudomyxoma peritonei.

Pseudomyxoma peritonei (PMP) is an indolent malignant syndrome. The standard treatment for PMP is cytoreductive surgery combined with intraperitoneal hyperthermic chemotherapy (CRS + HIPEC). However, the high recurrence rate and latent clinical symptoms and signs are major obstacles to further improving clinical outcomes. Moreover, patients in advanced stages receive little benefit from CRS + HIPEC due to widespread intraperitoneal metastases. Another challenge in PMP treatment involves the progressive sclerosis of PMP cell-secreted mucus, which is often increased due to activating mutations in the gene coding for guanine nucleotide-binding protein alpha subunit (GNAS). Consequently, the development of other PMP therapies is urgently needed. Several immune-related therapies have shown promise, including the use of bacterium-derived non-specific immunogenic agents, radio-immunotherapeutic agents, and tumor cell-derived neoantigens, but a well-recognized immunotherapy has not been established. In this review the roles of GNAS mutations in the promotion of mucin secretion and disease development are discussed. In addition, the immunologic features of the PMP microenvironment and immune-associated treatments are discussed to summarize the current understanding of key features of the disease and to facilitate the development of immunotherapies.

Mapping the complexity and diversity of tertiary lymphoid structures in primary and peritoneal metastatic gastric cancer.

BACKGROUND: Tertiary lymphoid structures (TLSs) are thought to stimulate antitumor immunity and positively impact prognosis and response to immune checkpoint blockade. In gastric cancers (GCs), however, TLSs are predominantly found in GC with poor prognosis and limited treatment response. We, therefore, hypothesize that immune cell composition and function of TLS depends on tumor location and the tumor immune environment. METHODS: Spatial transcriptomics and immunohistochemistry were used to characterize the phenotype of CD45+ immune cells inside and outside of TLS using archival resection specimens from GC primary tumors and peritoneal metastases. RESULTS: We identified significant intrapatient and interpatient diversity of the cellular composition and maturation status of TLS in GC. Tumor location (primary vs metastatic site) accounted for the majority of differences in TLS maturity, as TLS in peritoneal metastases were predominantly immature. This was associated with higher levels of tumor-infiltrating macrophages and Tregs and less plasma cells compared with tumors with mature TLS. Furthermore, mature TLSs were characterized by overexpression of antitumor immune pathways such as B cell-related pathways, MHC class II antigen presentation while immature TLS were associated with protumor pathways, including T cell exhaustion and enhancement of DNA repair pathways in the corresponding cancer. CONCLUSION: The observation that GC-derived peritoneal metastases often contain immature TLS which are associated with immune suppressive regulatory tumor-infiltrating leucocytes, is in keeping with the lack of response to immune checkpoint blockade and the poor prognostic features of peritoneal metastatic GC, which needs to be taken into account when optimizing immunomodulatory strategies for metastatic GC.

T-cell responses in colorectal peritoneal metastases are recapitulated in a humanized immune system mouse model.

Background: The occurrence of peritoneal metastasis (PM) in patients with colorectal cancer (CRC) has a dismal prognosis. There is often limited response to systemic- and immunotherapy, even in microsatellite unstable (MSI) CRC. To overcome therapy resistance, it is critical to understand local immune environment in the peritoneal cavity, and to develop models to study anti-tumor immune responses. Here, we defined the peritoneal immune system (PerIS) in PM-CRC patients and evaluate the pre-clinical potential of a humanized immune system (HIS) mouse model for PM-CRC. Methods: We studied the human PerIS in PM-CRC patients (n=20; MSS 19/20; 95%) and in healthy controls (n=3). HIS mice (NODscid gamma background; n=18) were generated, followed by intraperitoneal injection of either saline (HIS control; n=3) or human MSS/MSI CRC cell lines HUTU80, MDST8 and HCT116 (HIS-PM, n=15). Immune cells in peritoneal fluid and peritoneal tumors were analyzed using cytometry by time of flight (CyTOF). Results: The human and HIS mouse homeostatic PerIS was equally populated by NK cells and CD4+- and CD8+ T cells, however differences were observed in macrophage and B cell abundance. In HIS mice, successful peritoneal engraftment of both MSI and MSS tumors was observed (15/15; 100%). Both in human PM-CRC and in the HIS mouse PM-CRC model, we observed that MSS PM-CRC triggered a CD4+ Treg response in the PerIS, while MSI PM-CRC drives CD8+ TEMs responses. Conclusion: In conclusion, T cell responses in PM-CRC in HIS mice mirror those in human PM-CRC, making this model suitable to study antitumor T cell responses in PM-CRC.

Resiquimod-loaded cationic liposomes cure mice with peritoneal carcinomatosis and induce specific anti-tumor immunity.

Peritoneal carcinomatosis (PC) is characterized by a high recurrence rate and mortality following cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC), primarily due to incomplete cancer elimination. To enhance the standard of care for PC, we developed two cationic liposomal formulations aimed at localizing a toll-like receptor agonist, resiquimod (R848), in the peritoneal cavity to activate the immune system locally to specifically eradicate residual tumor cells. These formulations effectively extended R848 retention in the peritoneum by >10-fold, resulting in up to a 2-fold increase in interferon α (IFN-α) induction in the peritoneal fluid, without increasing the plasma levels. In a CT26 colon cancer model with peritoneal metastases, these liposomal R848 formulations, when combined with oxaliplatin (OXA)-an agent used in HIPEC that induces immunogenic cell death-increased tumor infiltration of effector immune cells, including DCs, CD4, and CD8 T cells. This led to the complete elimination of PC in 60-70% of the mice, while the control mice reached humane endpoints by 30 days. The cured mice developed specific antitumor immunity, as re-challenging them with the same tumor cells did not result in tumor establishment. However, inoculation with a different tumor line led to tumor development. Additionally, exposing CT26 tumor antigens to the splenocytes isolated from the cured mice induced the expansion of CD4 and CD8 T cells and the release of IFN-γ, demonstrating long-term immune memory to the specific tumor. The anti-tumor efficacy of these liposomal R848 formulations was mediated via CD8 T cells with different levels of involvement of CD4 and B cells, and the combination with an anti-PD-1 antibody achieved a cure rate of 90%.

Genomic and immune microenvironment features influencing chemoimmunotherapy response in gastric cancer with peritoneal metastasis: a retrospective cohort study.

BACKGROUND: Patients with peritoneal metastasis (PM) from gastric cancer (GC) exhibit poor prognosis. Chemoimmunotherapy offers promising clinical benefits; however, its efficacy and predictive biomarkers in a conversion therapy setting remain unclear. The authors aimed to retrospectively evaluate chemoimmunotherapy efficacy in a conversion therapy setting for GC patients with PM and establish a prediction model for assessing clinical benefits. MATERIALS AND METHODS: A retrospective evaluation of clinical outcomes encompassed 55 GC patients with PM who underwent chemoimmunotherapy in a conversion therapy setting. Baseline PM specimens were collected for genomic and transcriptomic profiling. Clinicopathological factors, gene signatures, and tumor immune microenvironment were evaluated to identify predictive markers and develop a prediction model. RESULTS: Chemoimmunotherapy achieved a 41.8% objective response rate and 72.4% R0 resection rate in GC patients with PM. Patients with conversion surgery showed better overall survival (OS) than those without the surgery (median OS: not reached vs 7.82 m, P <0.0001). Responders to chemoimmunotherapy showed higher ERBB2 and ERBB3 mutation frequencies, CTLA4 and HLA-DQB1 expression, and CD8+ T cell infiltration, but lower CDH1 mutation and naïve CD4+ T cell infiltration, compared to nonresponders. A prediction model was established integrating CDH1 and ERBB3 mutations, HLA-DQB1 expression, and naïve CD4+ T cell infiltration (AUC=0.918), which were further tested using an independent external cohort (AUC=0.785). CONCLUSION: This exploratory study comprehensively evaluated clinicopathological, genomic, and immune features and developed a novel prediction model, providing a rational basis for the selection of GC patients with PM for chemoimmunotherapy-involved conversion therapy.

A phase IA dose-escalation study of PHI-101, a new checkpoint kinase 2 inhibitor, for platinum-resistant recurrent ovarian cancer.

BACKGROUND: PHI-101 is an orally available, selective checkpoint kinase 2 (Chk2) inhibitor. PHI-101 has shown anti-tumour activity in ovarian cancer cell lines and impaired DNA repair pathways in preclinical experiments. Furthermore, the in vivo study suggests the synergistic effect of PHI-101 through combination with PARP inhibitors for ovarian cancer treatment. The primary objective of this study is to evaluate the safety and tolerability of PHI-101 in platinum-resistant recurrent ovarian cancer. METHODS: Chk2 inhibitor for Recurrent EpitheliAl periToneal, fallopIan, or oVarian cancEr (CREATIVE) trial is a prospective, multi-centre, phase IA dose-escalation study. Six cohorts of dose levels are planned, and six to 36 patients are expected to be enrolled in this trial. Major inclusion criteria include ≥ 19 years with histologically confirmed epithelial ovarian cancer, fallopian tube carcinoma, or primary peritoneal cancer. Also, patients who showed disease progression during platinum-based chemotherapy or disease progression within 24 weeks from completion of platinum-based chemotherapy will be included, and prior chemotherapy lines of more than five will be excluded. The primary endpoint of this study is to determine the dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of PHI-101. DISCUSSION: PHI-101 is the first orally available Chk2 inhibitor, expected to show effectiveness in treating recurrent ovarian cancer. Through this CREATIVE trial, DLT and MTD of this new targeted therapy can be confirmed to find the recommended dose for the phase II clinical trial. This study may contribute to developing a new combination regimen for the treatment of ovarian cancer. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04678102 .

5-aza-2'-deoxycytidine potentiates anti-tumor immunity in colorectal peritoneal metastasis by modulating ABC A9-mediated cholesterol accumulation in macrophages.

Background: 5-aza-2'-deoxycytidine (5Aza), a DNA methyltransferase (DNMT) inhibitor, could activate tumor adaptive immunity to inhibit tumor progression. However, the molecular mechanisms by which 5Aza regulates tumor immune microenvironment are still not fully understood. Methods: The role of 5Aza in immune microenvironment of peritoneal carcinomatosis (PC) of colorectal cancer (CRC) was investigated. The effects of 5Aza on macrophage activation were studied by flow cytometry, real-time PCR, Western blotting assays, and Drug Affinity Responsive Target Stability (DARTS). The effects of 5Aza on tumor immunity were validated in stromal macrophages and T cells from CRC patients. Results: 5Aza could stimulate the activation of macrophages toward an M1-like phenotype and subsequent activation of T cells in premetastatic fat tissues, and ultimately suppress CRC-PC in immune-competent mouse models. Mechanistically, 5Aza stimulated primary mouse macrophages toward to a M1-like phenotype characterized by the increase of p65 phosphorylation and IL-6 expression. Furthermore, we screened and identified ATP-binding cassette transporter A9 (ABC A9) as a binding target of 5Aza. 5Aza induced cholesterol accumulation, p65 phosphorylation and IL-6 expression in an ABC A9-dependent manner. Pharmacological inhibition of NF-κB, or genetic depletion of IL-6 abolished the antitumor effect of 5Aza in mice. In addition, the antitumor effect of 5Aza was synergistically potentiated by conventional chemotherapeutic drugs 5-Fu or OXP. Finally, we validated the reprogramming role of 5Aza in antitumor immunity in stromal macrophages and T cells from CRC patients. Conclusions: Taken together, our findings showed for the first time that 5Aza suppressed CRC-PC by regulating macrophage-dependent T cell activation in premetastatic microenvironment, meanwhile uncovered a DNA methylation-independent mechanism of 5Aza in regulating ABC A9-associated cholesterol metabolism and macrophage activation.

Inflammatory macrophages exploit unconventional pro-phagocytic integrins for phagocytosis and anti-tumor immunity.

Blockade of the inhibitory checkpoint SIRPα-CD47 promotes phagocytosis of cancer cells by macrophages and is a promising avenue in anti-cancer therapy. Productive phagocytosis is strictly predicated on co-engagement of pro-phagocytic receptors-namely, Fc receptors (FcRs), integrin CD11b, or SLAMF7-by their ligands on cancer cells. Here, we examine whether additional pro-phagocytic receptors could be harnessed to broaden the scope of phagocytosis. Inflammatory stimuli, including multiple cytokines and Toll-like receptor (TLR) ligands, augment phagocytosis efficiency and fully alleviate the requirement of FcRs, CD11b, and SLAMF7 for phagocytosis. These effects are mediated by the unconventional pro-phagocytic integrins CD11a and CD11c, which act with CD18 to initiate actin polarization, leading to phagocytosis. Some inflammatory stimuli enable phagocytosis even in the absence of SIRPα-CD47 blockade. Higher CD11c expression in macrophage-enriched tumors correlates with improved survival in clinical studies. Thus, inflammatory macrophages exploit unconventional pro-phagocytic integrins for improved phagocytosis and anti-tumor immunity.

Unconventional radiotherapy to enhance immunotherapy efficacy in bulky tumors: a case report.

Determining the most appropriate management strategy for patients with large tumor masses is a very challenging issue. Unconventional radiotherapy modalities, such as spatially fractionated radiation therapy (SFRT), are associated with dramatic responses. Recent studies have suggested that systemic immune activation may be triggered by SFRT delivery to primary tumor lesion. This report describes the case of a patient treated with a novel form of immune-sparing partially ablative irradiation (ISPART) for a bulky peritoneal metastasis from renal cell cancer, refractory to anti-PD-1 therapy (nivolumab) as third-line therapy after sequential therapy with sunitinib and cabozantinib. The observed response suggests that there may be a synergistic effect between ISPART and immunotherapy. This case report supports the inclusion of ISPART in patients presenting with bulky lesions treated with checkpoint inhibitors .

Lay abstract Managing large tumor masses is a very challenging issue. In recent years, radiotherapy methods have been linked with good responses, which may be due to the activation of immune mechanisms. We describe the case of a patient with a large tumor mass from renal cell cancer. The patient had already been treated with anti-PD-1 therapy, after treatment with sunitinib and cabozantinib, along with radiotherapy. The results suggest that radiotherapy together with immunotherapy is very effective in enhancing immune response.

The association of immunosurveillance and distant metastases in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the third most common malignancy worldwide, but the key driver to distant metastases is still unknown. This study aimed to elucidate the link between immunosurveillance and organotropism of metastases in CRC by evaluating different gene signatures and pathways. MATERIAL AND METHODS: CRC patients undergoing surgery at the Department of General, Visceral and Transplantation Surgery at the Ludwig-Maximilian University Hospital Munich (Munich, Germany) were screened and categorized into M0 (no distant metastases), HEP (liver metastases) and PER (peritoneal carcinomatosis) after a 5-year follow-up. Six patients of each group were randomly selected to conduct a NanoString analysis, which includes 770 genes. Subsequently, all genes were further analyzed by gene set enrichment analysis (GSEA) based on seven main cancer-associated databases. RESULTS: Comparing HEP vs. M0, the gene set associated with the Toll-like receptor (TLR) cascade defined by the Reactome database was significantly overrepresented in HEP. HSP90B1, MAPKAPK3, PPP2CB, PPP2R1A were identified as the core enrichment genes. The immunologic signature pathway GSE6875_TCONV_VS_FOXP3_KO_TREG_DN with FOXP3 as downstream target was significantly overexpressed in M0. RB1, TMEM 100, CFP, ZKSCAN5, DDX50 were the core enrichment genes. Comparing PER vs. M0 no significantly differentially expressed gene signatures were identified. CONCLUSION: Chronic inflammation might enhance local tumor growth. This is the first study identifying immune related gene sets differentially expressed between patients with either liver or peritoneal metastases. The present findings suggest that the formation of liver metastases might be associated with TLR-associated pathways. In M0, a high expression of FOXP3 + tumor infiltrating lymphocytes (TILs) seemed to prevent at least in part metastases. Thus, these correlative findings lay the cornerstone to further studies elucidating the underlying mechanisms of organotropism of metastases.

TGFBI Production by Macrophages Contributes to an Immunosuppressive Microenvironment in Ovarian Cancer.

The tumor microenvironment evolves during malignant progression, with major changes in nonmalignant cells, cytokine networks, and the extracellular matrix (ECM). In this study, we aimed to understand how the ECM changes during neoplastic transformation of serous tubal intraepithelial carcinoma lesions (STIC) into high-grade serous ovarian cancers (HGSOC). Analysis of the mechanical properties of human fallopian tubes (FT) and ovaries revealed that normal FT and fimbria had a lower tissue modulus, a measure of stiffness, than normal or diseased ovaries. Proteomic analysis of the matrisome fraction between FT, fimbria, and ovaries showed significant differences in the ECM protein TGF beta induced (TGFBI, also known as ßig-h3). STIC lesions in the fimbria expressed high levels of TGFBI, which was predominantly produced by CD163-positive macrophages proximal to STIC epithelial cells. In vitro stimulation of macrophages with TGFß and IL4 induced secretion of TGFBI, whereas IFNγ/LPS downregulated macrophage TGFBI expression. Immortalized FT secretory epithelial cells carrying clinically relevant TP53 mutations stimulated macrophages to secrete TGFBI and upregulated integrin αvß3, a putative TGFBI receptor. Transcriptomic HGSOC datasets showed a significant correlation between TGFBI expression and alternatively activated macrophage signatures. Fibroblasts in HGSOC metastases expressed TGFBI and stimulated macrophage TGFBI production in vitro. Treatment of orthotopic mouse HGSOC tumors with an anti-TGFBI antibody reduced peritoneal tumor size, increased tumor monocytes, and activated ß3-expressing unconventional T cells. In conclusion, TGFBI may favor an immunosuppressive microenvironment in STICs that persists in advanced HGSOC. Furthermore, TGFBI may be an effector of the tumor-promoting actions of TGFß and a potential therapeutic target. SIGNIFICANCE: Analysis of ECM changes during neoplastic transformation reveals a role for TGFBI secreted by macrophages in immunosuppression in early ovarian cancer.

Effect of Preoperative Immunonutrition on Postoperative Major Morbidity after Cytoreductive Surgery and HIPEC in Patients with Peritoneal Metastasis.

The effect of preoperative immunonutrition intake on postoperative major complications in patients following cytoreductive surgery (CRS) with or without hyperthermic intraperitoneal chemotherapy (HIPEC) was assessed. The accuracy of C-Reactive Protein (CRP) for detecting postoperative complications was also analyzed. Patients treated within a peritoneal carcinomatosis program in which a complete or optimal cytoreduction was achieved were retrospectively analyzed. They were divided into two groups based on whether preoperative immunonutrition (IMN) or not (non-IMN) were administered. Clinical and surgical variables and postoperative complications were gathered. Predictive values of major morbidity of CRP during the first 3 postoperative days (POD) were also evaluated. A total of 107 patients were included, 48 belonging to the IMN group and 59 to the non-IMN group. In multivariate analysis immunonutrition (OR 0.247; 95%CI 0.071-0.859; p = 0.028), and the number of visceral resections (OR 1.947; 95%CI 1.086-3.488; p = 0.025) emerged as independent factors associated with postoperative major morbidity. CRP values above 103 mg/L yielded a negative predictive value of 84%. Preoperative intake of immunonutrition was associated with a decrease of postoperative major morbidity and might be recommended to patients with peritoneal carcinomatosis following CRS. Measuring CRP levels during the 3 first postoperative days is useful to rule out major morbidity.

Abundant intratumoral fibrosis prevents lymphocyte infiltration into peritoneal metastases of colorectal cancer.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) have been reported to reflect the anti-tumor immune status. However, recent investigations have demonstrated that intratumoral fibrosis is important as a factor affecting the infiltration of TILs. This study investigated the organ specificities of TIL infiltration and intratumoral fibrosis in primary colorectal cancer and distant metastases, as well as the relationship between the distribution of TILs and intratumoral fibrosis. METHODS: Patients who underwent resection of primary tumors or distant metastases for colorectal cancer with distant metastases were enrolled. We evaluated the TIL infiltration by immunohistochemical staining with CD3&CD8 and intratumoral fibrosis by immunohistochemical staining with α-SMA positive cancer-associated fibroblasts and Masson's trichrome staining against collagen fibers. The "ImageJ" was used to evaluate fibrosis, and the density of TILs in the dense and sparse areas of fibrosis was calculated. The Immunoscore (IS) was obtained based on the density of CD3+/CD8+TILs in the tumor center and invasive margin of the primary tumor. RESULTS: The degree of CD3+/CD8+TIL infiltration in peritoneal metastases was significantly lower than that in liver and lung metastases. The area ratio of α-SMA positive cancer-associated fibroblasts and collagen fibers in peritoneal metastases was significantly higher than that of liver and lung metastases. Furthermore, the density of TILs in the high-fibrosis area was significantly lower than that in the low-fibrosis area. In the high-IS group of primary tumors, the degree of TIL infiltration in distant metastases was significantly higher than that in the low-IS group. CONCLUSION: The infiltration of T lymphocytes into tumors is prevented in peritoneal metastases of colorectal cancer due to the high intratumoral fibrosis, which may lead to treatment resistance and a poor prognosis.

SENTI-101, a Preparation of Mesenchymal Stromal Cells Engineered to Express IL12 and IL21, Induces Localized and Durable Antitumor Immunity in Preclinical Models of Peritoneal Solid Tumors.

Advanced peritoneal carcinomatosis including high-grade ovarian cancer has poor prognoses and a poor response rate to current checkpoint inhibitor immunotherapies; thus, there is an unmet need for effective therapeutics that would provide benefit to these patients. Here we present the preclinical development of SENTI-101, a cell preparation of bone marrow-derived mesenchymal stromal (also known as stem) cells (MSC), which are engineered to express two potent immune-modulatory cytokines, IL12 and IL21. Intraperitoneal administration of SENTI-101 results in selective tumor-homing and localized and sustained cytokine production in murine models of peritoneal cancer. SENTI-101 has extended half-life, reduced systemic distribution, and improved antitumor activity when compared with recombinant cytokines, suggesting that it is more effective and has lower risk of systemic immunotoxicities. Treatment of tumor-bearing immune-competent mice with a murine surrogate of SENTI-101 (mSENTI-101) results in a potent and localized immune response consistent with increased number and activation of antigen presenting cells, T cells and B cells, which leads to antitumor response and memory-induced long-term immunity. Consistent with this mechanism of action, co-administration of mSENTI-101 with checkpoint inhibitors leads to synergistic improvement in antitumor response. Collectively, these data warrant potential clinical development of SENTI-101 for patients with peritoneal carcinomatosis and high-grade ovarian cancer.Graphical abstract: SENTI-101 schematic and mechanism of actionSENTI-101 is a novel cell-based immunotherapeutic consisting of bone marrow-derived mesenchymal stromal cells (BM-MSC) engineered to express IL12 and IL21 intended for the treatment of peritoneal carcinomatosis including high-grade serous ovarian cancer. Upon intraperitoneal administration, SENTI-101 homes to peritoneal solid tumors and secretes IL12 and IL21 in a localized and sustained fashion. The expression of these two potent cytokines drives tumor infiltration and engagement of multiple components of the immune system: antigen-presenting cells, T cells, and B cells, resulting in durable antitumor immunity in preclinical models of cancer.

Immune Landscape of Gastric Carcinoma Tumor Microenvironment Identifies a Peritoneal Relapse Relevant Immune Signature.

Background: Gastric cancer (GC) still represents the third leading cause of cancer-related death worldwide. Peritoneal relapse (PR) is the most frequent metastasis occurring among patients with advanced gastric cancer. Increasingly more evidence have clarified the tumor immune microenvironment (TIME) may predict survival and have clinical significance in GC. However, tumor-transcriptomics based immune signatures derived from immune profiling have not been established for predicting the peritoneal recurrence of the advanced GC. Methods: In this study, we depict the immune landscape of GC by using transcriptome profiling and clinical characteristics retrieved from GSE62254 of Gene Expression Omnibus (GEO). Immune cell infiltration score was evaluated via single-sample gene set enrichment (ssGSEA) analysis algorithm. The least absolute shrinkage and selection operator (LASSO) Cox regression algorithm was used to select the valuable immune cells and construct the final model for the prediction of PR. The receiver operating characteristic (ROC) curve and the Kaplan-Meier curve were used to check the accuracy of PRIs. Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to explore the molecular pathways associated with PRIs. Results: A peritoneal recurrence related immune score (PRIs) with 10 immune cells was constructed. Compared to the low-PRIs group, the high-PRIs group had a greater risk. The upregulation of the focal adhesion signaling was observed in the high-PRIs subtype by GSEA and KEGG. Multivariate analysis found that both in the internal training cohort and the internal validation cohort, PRIs was a stable and independent predictor for PR. A nomogram that integrated clinicopathological features and PRIs to predict peritoneal relapse was constructed. Subgroup analysis indicated that the PRIs could obviously distinguish peritoneal recurrence in different molecular subtypes, pathological stages and Lauren subtypes, in which PRIs of Epithelial-Mesenchymal Transitions (EMT) subtype, III-IV stage and diffuse subtype are higher respectively. Conclusion: Overall, we performed a comprehensive evaluation of the immune landscape of GC and constructed a predictive PR model based on the immune cell infiltration. The PRIs represents novel promising feature of predicting peritoneal recurrence of GC and sheds light on the improvement of the personalized management of GC patients after surgery.

Identification of novel autoantibodies in ascites of relapsed paclitaxel-resistant gastric cancer with peritoneal metastasis using immunome protein microarrays and proteomics.

BACKGROUND: Gastric cancer (GC) patients with peritoneal metastasis usually have extremely poor prognosis. Intraperitoneal infusion of paclitaxel (PTX) provides an effective treatment, but relapse and PTX-resistance are unavoidable disadvantages, and it is difficult to monitor the occurrence of PTX-resistance. OBJECTIVE: The aim of this study was to explore novel autoantibodies in the ascites of individuals with relapsed PTX-resistant GC with peritoneal metastasis. METHODS: Ascites samples were collected before PTX infusion and after the relapse in 3 GC patients. To determine the expression of significantly changed proteins, we performed autoantibody profiling with immunome protein microarrays and tandem mass tag (TMT) quantitative proteomics, and then, the overlapping proteins were selected. RESULTS: Thirty-eight autoantibodies that were differentially expressed between the ascites in the untreated group and relapsed PTX-resistant group were identified. For confirmation of the results, TMT quantitative proteomics was performed, and 842 dysregulated proteins were identified. Four proteins, TPM3, EFHD2, KRT19 and vimentin, overlapped between these two assays. CONCLUSIONS: Our results first revealed that TPM3, EFHD2, KRT19 and vimentin were novel autoantibodies in the ascites of relapsed PTX-resistant GC patients. These autoantibodies may be used as potential biomarkers to monitor the occurrence of PTX-resistance.

High CD8/CD33 ratio in peritoneal metastatic lesions is associated with favorable prognosis in gastric cancer.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) and other immune cells have been reported as a prognostic factor in several tumors, including gastric cancer, and they play an important role in antitumor effect at the primary site. There were few reports on the immune status in peritoneal metastatic lesions for gastric cancer. AIMS: The aims of this study were to assess the prognostic significance of TILs (CD4, CD8, CD19, regulatory T cells [Tregs]), and myeloid-derived suppressor cells (MDSCs) in peritoneal metastatic lesions. METHODS: We retrospectively investigated 60 patients for gastric cancer with peritoneal metastasis who were treated between 2009 and 2016 in our institute. Immunohistochemistry for CD4, CD8, CD19, FOXP3, and CD33 was performed in the peritoneal metastatic lesions. The absolute numbers of immune cells and ratios were evaluated, and the relationship between immune-related marker and overall survival (OS) was investigated. RESULTS: A high infiltration of CD8+ lymphocytes or high CD8/CD33 ratio was a better prognosis for OS in univariate analysis using all immunologic variables (P = .012, P = .001). In multivariate analysis for clinical and immunologic variables, high CD8/CD33 ratio was identified as an independent prognostic factor for OS (Hazard ratio: 0.291, 95% confidence interval: 0.126-0.670, P = .004). CONCLUSION: High CD8/CD33 ratio and high infiltration of CD8+ lymphocytes in peritoneal metastatic lesions were favorable prognoses for gastric cancer patients with peritoneal metastasis. It is necessary to modify the immune microenvironment result to increase the level of CD8+ lymphocytes in the peritoneal metastatic lesions.

Natural killer T cell immunotherapy combined with oncolytic vesicular stomatitis virus or reovirus treatments differentially increases survival in mouse models of ovarian and breast cancer metastasis.

BACKGROUND: Oncolytic viruses reduce tumor burden in animal models and have generated promising results in clinical trials. However, it is likely that oncolytic viruses will be more effective when used in combination with other therapies. Current therapeutic approaches, including chemotherapeutics, come with dose-limiting toxicities. Another option is to combine oncolytic viruses with immunotherapeutic approaches. METHODS: Using experimental models of metastatic 4T1 breast cancer and ID8 ovarian peritoneal carcinomatosis, we examined natural killer T (NKT) cell-based immunotherapy in combination with recombinant oncolytic vesicular stomatitis virus (VSV) or reovirus. 4T1 mammary carcinoma cells or ID8 ovarian cancer cells were injected into syngeneic mice. Tumor-bearing mice were treated with VSV or reovirus followed by activation of NKT cells via the intravenous administration of autologous dendritic cells loaded with the glycolipid antigen α-galactosylceramide. The effects of VSV and reovirus on immunogenic cell death (ICD), cell viability and immunogenicity were tested in vitro. RESULTS: VSV or reovirus treatments followed by NKT cell activation mediated greater survival in the ID8 model than individual therapies. The regimen was less effective when the treatment order was reversed, delivering virus treatments after NKT cell activation. In the 4T1 model, VSV combined with NKT cell activation increased overall survival and decreased metastatic burden better than individual treatments. In contrast, reovirus was not effective on its own or in combination with NKT cell activation. In vitro, VSV killed a panel of tumor lines better than reovirus. VSV infection also elicited greater increases in mRNA transcripts for proinflammatory cytokines, chemokines, and antigen presentation machinery compared with reovirus. Oncolytic VSV also induced the key hallmarks of ICD (calreticulin mobilization, plus release of ATP and HMGB1), while reovirus only mobilized calreticulin. CONCLUSION: Taken together, these results demonstrate that oncolytic VSV and NKT cell immunotherapy can be effectively combined to decrease tumor burden in models of metastatic breast and ovarian cancers. Oncolytic VSV and reovirus induced differential responses in our models which may relate to differences in virus activity or tumor susceptibility.

Cancer-derived Exosomes Activate Immune Surveillance and Suppress Peritoneal Metastasis of Murine Colonic Cancer.

BACKGROUND: Colonic cancer is associated with a low incidence of peritoneal metastasis compared with gastric cancer; however, the reason for this remains unclear. In this study, a model of peritoneal dissemination using the CT26 murine colon cancer cell line was used to analyze the physiological roles of cancer-derived exosomes. MATERIALS AND METHODS: Exosomes were collected from the supernatant of CT26 cell culture by ultracentrifugation. The number of peritoneal disseminations in two mouse models of colonic cancer pre-administered exosomes or phosphate-buffered saline were compared. RESULTS: Cancer-derived exosomes suppressed peritoneal dissemination compared to phosphate-buffered saline. After administration of exosomes, the number of intraperitoneal macrophages and the expression of inducible nitric oxide synthase increased. Furthermore, cancer-derived exosomes increased activated natural killer cells and interferon-γ expression. CONCLUSION: Tumor-derived exosomes from colonic cancer may suppress peritoneal metastasis via an immunological mechanism.