Cellular Immunology

Exosomes derived from ovarian cancer promote the progression of ovarian cancer through macrophage M2 polarization mediated by the THBS1/TGFBI signaling axis

Tumor-derived exosomes play a critical role in facilitating intercellular communication between cancer cells and tumor-associated macrophages (TAMs). Nevertheless, the precise molecular mechanisms underlying exosome-mediated interactions specifically in ovarian cancer remain incompletely elucidated. TAMs were treated with exosomes isolated from clinical ovarian cancer specimens. Macrophage polarization was assessed using qRT-PCR, and western blot analysis. RNA sequencing was employed to identify key genes within the exosomes. The malignant phenotype of ovarian cancer cells was evaluated through cell counting kit-8 (CCK-8), Transwell assay, and wound-healing assays. Our findings showed that exosomes derived from both early and late-stage malignant ovarian cancer tissues induced the upregulation of all M2 macrophage markers and the downregulation of M1 markers. RNA sequencing analysis identified thrombospondin-1 (THBS1) as a potential pivotal gene influencing exosome-regulated TAM polarization. THBS1 knockdown within exosomes inhibited the polarization of TAMs toward the M2 phenotype and concurrently decreased transforming growth factor beta induced (TGFBI) expression in macrophages. Notably, TGFBI knockdown in TAM reversed the M2 polarization induced by ovarian cancer cells-derived exosomes. In vivo, ovarian cancer cell-derived exosomes facilitate cancer progression, concomitantly increasing the polarization of M2 macrophages and upregulating THBS1 and TGFBI expression within tumor tissues. THBS1, carried by ovarian cancer-derived exosomes, promotes M2 polarization of TAMs by modulating TGFBI expression. The subsequent M2 polarization of TAMs contributes to the establishment of an immunosuppressive tumor microenvironment, thereby facilitating disease progression. Consequently, targeting the exosome-mediated signaling axis between cancer cells and macrophages represents a promising avenue for developing novel therapeutic interventions.

EGCG remodels the TGF-β cervical cancer micro-environment towards immune responsiveness

Exploring the role of immune modulators alongside TGF-β in cervical cancer (CC) and PBMCs may improve the understanding of targeted treatment strategies. We analyzed expression, overall survival (OS), correlation and tumor infiltration of PD-L1, CD55 and CD46, as well as immune cell fractions in CC patients using OncoDB, TIMER 2.0 and TCIA. RT-PCR and western blotting was performed to assess PD-L1, CD55 and CD46 expression. Viability, mitored, apoptosis and MMP-2 were evaluated in CC cells co-cultured with PBMCs. Morphology, crystal violet staining, ROS and MMP-2 were examined in SiHa spheroids. PD-L1 was upregulated, CD55 was significantly increased and CD46 showed no significant difference in HPV16 positive compared with HPV negative individuals. Elevated PD-L1, CD55 and CD46 were associated with reduced OS in HPV16 positive individuals. PD-L1 and CD55 showed moderate positive and negative correlation with TGF-β, respectively, whereas CD46 correlation was negligible. Immune fractions including M1 macrophages (31 %), M2 macrophages (17 %), CD8 T cells (21 %), NK cells (10 %), were linked to reduced OS. TGF-β, PD-L1 and CD46 infiltration were positively corelated with CD8+ T cells. In CC cells, TGF-β stimulation increased PD-L1, while decreasing CD55 and CD46, reducing viability, metabolic activity and inducing apoptosis in HPV (+) co-cultures. EGCG treatment under TGF-β, reduced PD-L1, CD55 and CD46 expression, decreased viability, metabolic activity and MMP-2 secretion, while inducing apoptosis in SiHa co-culture. In 3D spheroids, EGCG inhibited proliferation and MMP-2 activity while increasing ROS production. EGCG, by targeting TGF-β and modulating PD-L1 and mCRPs, represents a promising candidate for immunotherapeutic development in CC.

A synergy of CD133 overexpression and TGF-β supplementation in tumorigenesis of ovarian cancer cell lines in a three-dimensional sphere forming model

Ovarian cancer (OC) is a highly lethal gynecological malignancy, mainly due to chemoresistance and tumor recurrence. Cancer stem cells (CSCs) may be responsible for chemoresistance, and CSC has become a new target for treatment. In this study, we aimed to develop a three-dimensional (3D) OC model with well-recapitulated stemness in the tumor microenvironment (TME). We observed that the niche-like environment associated with CSC properties is characterized by the presence of CD133-positive cells during OC sphere induction. The cancer-associated fibroblast (CAF)-integrated 3D multicellular OC model recapitulates enhanced tumorigenicity and cytokine-mediated invasiveness more than the 2D monolayer culture. Chemoresistance of the 3D OC model is also acquired. In addition, the in vivo growth of an established xenograft model with a 3D CAF-integrated OC sphere exhibits proper stemness features and full cancer-associated markers for tumorigenesis. After transduction of the CD133 gene into OC cells, gene ontology (GO) and KEGG pathway enrichment analyses reveal that cytokine-mediated endothelial mesenchymal transition (EMT) is possibly responsible for chemotherapy resistance and tumor progression, and enhanced PAR1, CXCR4, and PD-L1 expressions are also observed. In addition, we found that engineered chimeric antigen receptor (CAR)-T cells targeting PAR1 demonstrated significant in vitro cytotoxicity toward chemoresistant OC sphere with CD133 overexpression. Taken together, our results show that a CD133-3D OC sphere recaptures TME that mimics a real late-stage OC condition, and it can act as a useful platform with mechanism-verifying in vitro and in vivo experiments in researching OC chemotherapy, immunotherapy, and cell therapy to discover new therapeutic approaches.

KLHDC8A knockdown in normal ovarian epithelial cells promoted the polarization of pro-tumoral macrophages via the C5a/C5aR/p65 NFκB signaling pathway

Tumor-associated macrophages (TAM) is related to Ovarian cancer (OC) pathogenesis, but the exact mechanism remains unclear. This study investigated the expression of Kelch Domain Containing 8 A (KLHDC8A) in OC and the mechanism associated with TAM. Bioinformatics analysis of differential expression genes between normal and OC tissues were analyzed based on the Tumor Genome Atlas (TCGA) databases. KLHDC8A mRNA expression was knocked down in normal epithelial cells (IOSE80), and then the effects of siKLHDC8A on the proliferation, invasion, migration and C5a secretion of IOSE80 cells were explored. THP1-derived macrophages were cultured with medium of NC-IOSE80 cells, siKLHDC8A-IOSE80 cells with or without C5aR antagonists. KLHDC8A was lowly expressed in OC and negatively correlated with the infiltration of tumor-promoting macrophages, contributing to the survival of OC patients. Furthermore, siKLHDC8A promotes the proliferation, invasion and migration of IOSE80 cells and leads to polarization of pro-tumoral macrophages, which can be rescued by C5aR antagonists. Our results indicated that KLHDC8A knockdown could modulate the development of OC by affecting macrophage polarization to pro-tumoral type via the C5a/C5aR/p65 NFκB signaling pathway. It may play an essential role as the tumor suppressor genes in diagnosis and treatment of OC.

Loss of B1 and marginal zone B cells during ovarian cancer

Recent advances in immunotherapy have not addressed the challenge presented by ovarian cancer. Although the peritoneum is an "accessible" locus for this disease there has been limited characterization of the immunobiology therein. We investigated the ID8-C57BL/6J ovarian cancer model and found marked depletion of B1 cells from the ascites of the peritoneal cavity. There was also selective loss of the B1 and marginal zone B cell subsets from the spleen. Immunity to antigens that activate these subsets validated their loss rather than relocation. A marked influx of myeloid-derived suppressor cells correlated with B cell subset depletion. These observations are discussed in the context of the housekeeping burden placed on innate B cells during ovarian cancer and to foster consideration of B cell biology in therapeutic strategies to address this challenge.

In vitro ovarian tumor-conditioned CD163+ human macrophages retain phagocytic response to CD47 blockade

CD163-expressing macrophages are abundant in ovarian cancer where they accelerate tumor growth and metastasis. CD47 blockade is a novel immunotherapy aiming to activate macrophage phagocytosis of tumor cells, but it is currently unknown if the tumor-associated macrophages expressing CD163 respond poorly to CD47 blockade. Human monocyte-derived macrophages were exposed to tumor-conditioned medium from A2780 ovarian cancer cells during differentiation. Effects on gene expression, membrane protein levels, release of soluble proteins and macrophage phagocytosis of A2780 cells in response to CD47 blockade were measured and compared to control macrophages. Tumor cell conditioning induced macrophage expression of CD163 on both the mRNA and protein level. Furthermore, tumor conditioning simultaneously increased protein expression of the phenotype markers CD206 and CD80, and the phagocytosis checkpoint LILRB1. However, tumor conditioning did not reduce phagocytic capacity, as CD47 blockade induced macrophage phagocytosis of A2780 cells to similar degrees in both control and tumor cell-conditioned macrophages. In vitro tumor conditioning did not reduce the phagocytic response to CD47 blockade, suggesting that induction of a macrophage phenotype with increased expression of CD163 does not directly limit the capacity for phagocytosis of tumor cells. In conclusion, these findings suggest that CD163+ macrophages remain responsive to CD47 blockade, highlighting their potential as targets for immunotherapy in ovarian cancer.