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Ovarian cancer metastasis occurs primarily in the peritoneal cavity. Orchestration of cancer cells with various cell types, particularly macrophages, in the peritoneal cavity creates a metastasis-favorable environment. In the past decade, macrophage heterogeneities in different organs as well as their diverse roles in tumor settings have been an emerging field. This review highlights the unique microenvironment of the peritoneal cavity, consisting of the peritoneal fluid, peritoneum, and omentum, as well as their own resident macrophage populations. Contributions of resident macrophages in ovarian cancer metastasis are summarized; potential therapeutic strategies by targeting such cells are discussed. A better understanding of the immunological microenvironment in the peritoneal cavity will provide a stepping-stone to new strategies for developing macrophage-based therapies and is a key step toward the unattainable eradication of intraperitoneal metastasis of ovarian cancer.

Aberrant glycosylation is a crucial strategy employed by cancer cells to evade cellular immunity. However, it’s unclear whether homologous recombination (HR) status-dependent glycosylation can be therapeutically explored. Here, we show that the inhibition of branched N -glycans sensitizes HR-proficient, but not HR-deficient, epithelial ovarian cancers (EOCs) to immune checkpoint blockade (ICB). In contrast to fucosylation whose inhibition sensitizes EOCs to anti-PD-L1 immunotherapy regardless of HR-status, we observe an enrichment of branched N -glycans on HR-proficient compared to HR-deficient EOCs. Mechanistically, BRCA1/2 transcriptionally promotes the expression of MGAT5, the enzyme responsible for catalyzing branched N -glycans. The branched N -glycans on HR-proficient tumors augment their resistance to anti-PD-L1 by enhancing its binding with PD-1 on CD8 + T cells. In orthotopic, syngeneic EOC models in female mice, inhibiting branched N -glycans using 2-Deoxy-D-glucose sensitizes HR-proficient, but not HR-deficient EOCs, to anti-PD-L1. These findings indicate branched N -glycans as promising therapeutic targets whose inhibition sensitizes HR-proficient EOCs to ICB by overcoming immune evasion. Cancer cells can employ aberrant glycosylation patterns to evade the host immune response. Here the authors report that inhibition of branched N-glycans sensitizes homologous recombination (HR)-proficient, but not HR-deficient, epithelial ovarian cancer to immune checkpoint inhibitors.

Availability of oxygen plays an important role in tissue organization and cell-type specific metabolism. It is, however, difficult to analyze hypoxia-related adaptations in vitro because of inherent limitations of experimental model systems. In this study, we establish a microfluidic tissue culture protocol to generate hypoxic gradients in vitro, mimicking the conditions found in the liver acinus. To accomplish this, four microfluidic chips, each containing two chambers, were serially connected to obtain eight interconnected chambers. HepG2 hepatocytes were uniformly seeded in each chamber and cultivated under a constant media flow of 50 µL/h for 72 h. HepG2 oxygen consumption under flowing media conditions established a normoxia to hypoxia gradient within the chambers, which was confirmed by oxygen sensors located at the inlet and outlet of the connected microfluidic chips. Expression of Hif1α mRNA and protein was used to indicate hypoxic conditions in the cells and albumin mRNA and protein expression served as a marker for liver acinus-like zonation. Oxygen measurements performed over 72 h showed a change from 17.5% to 15.9% of atmospheric oxygen, which corresponded with a 9.2% oxygen reduction in the medium between chamber1 (inlet) and 8 (outlet) in the connected microfluidic chips after 72 h. Analysis of Hif1α expression and nuclear translocation in HepG2 cells additionally confirmed the hypoxic gradient from chamber1 to chamber8. Moreover, albumin mRNA and protein levels were significantly reduced from chamber1 to chamber8, indicating liver acinus zonation along the oxygen gradient. Taken together, microfluidic cultivation in interconnected chambers provides a new model for analyzing cells in a normoxic to hypoxic gradient in vitro. By using a well-characterized cancer cell line as a homogenous hepatocyte population, we also demonstrate that an approximate 10% reduction in oxygen triggers translocation of Hif1α to the nucleus and reduces albumin production.

Over recent decades, stereotactic body radiotherapy has garnered increasing popularity. Unfortunately, conventional preclinical 2D in vitro models are often insufficient for studying radiotherapy effects. Therefore, in this study, we developed a novel anthropomorphic in vitro liver phantom, which simulates the relevant hepatocellular carcinoma (HCC) tumor microenvironment and spatial organization. The liver phantom was 3D printed, filled with tissue-mimicking agarose mixture, and designed to fit ten microfluidic chips (MCs), in which HepG2 cells were seeded. Airtight MCs induced hypoxic conditions, as verified by Hif1α staining. Irradiation was conducted with 20 Gy in one fraction using a CyberKnife, in either a 2D setup, or by irradiating MCs arranged in the 3D-printed liver model using an individually calculated treatment plan. Post-irradiation cellular damage was determined via γH2AX staining. Here, we demonstrate a new physiologically relevant approach to model HCC pathology following radiotherapy. Comparing γH2AX staining in normoxic conditions to cells grown in MCs (hypoxic conditions) revealed a reduction in cellular damage of 30.24% (p = 0.0001) in the hypoxic environment. Moreover, we compared the scattering effect of radiation on a conventional 2D in vitro model to our new 3D anthropomorphic liver phantom and observed a significant γH2AX intensity reduction of 9.6% (p = 0.0294) in HepG2 cells irradiated in the phantom. Our approach of utilizing a liver phantom takes into account the hypoxic tumor microenvironment and 3D scattering effects of tissue irradiation, thereby modeling both physical and biological parameters of HCC tumors. The use of tissue phantoms lays the groundwork for future examination of other hypoxic tumors and offers a more comprehensive approach for screening and analysis of novel cancer therapeutics.

BackgroundOvarian cancer (OvCa) is the most lethal gynecological cancer and the fifth leading cause of cancer-related deaths in women. Despite an initial positive response to therapy, most patients relapse and present with chemotherapy resistance. The prognosis for recurrent disease is poor, with a 5-year survival rate of < 30%. One contributor to disease recurrence and therapy resistance is the presence of disseminated cancer cells that remain in the peritoneal fluid after treatment. A growing body of evidence suggests that these cells exhibit dormancy characteristics that render them resistant to most therapies. Moreover, cancer cells in fluid are unable to be surgically resected. Because 75% of relapsed patients present with therapy-resistant intraperitoneal disease, developing new strategies to effectively target disseminating OvCa cells in the peritoneal fluid is crucial for the effective treatment of OvCa. Two key players in peritoneal immunity, the omentum and peritoneal resident macrophages (PRMΦs), are known to sequester pathogens in the peritoneal fluid and coordinate an inflammatory immune response in the peritoneum. One such activator of peritoneal immunity is β-glucan, a sugar found on the cell walls of yeasts.ObjectiveTo target disseminating OvCa cells in the peritoneal fluid by activating myeloid cells in the peritoneal cavity via intraperitoneal administration of β-glucan.MethodsC57bl/6J mice were injected with GFP-labeled murine OvCa cells (KPCA: Trp53 −/−R172H Ccne1 OE Akt2 OE KRAS G12V) immediately followed by 500μg β-glucan (i.p.). Five hours later, mice were euthanized and their peritoneal lavage was analyzed for the presence of OvCa cells. To model advanced disease, Luciferase-KPCA cells were seeded in C57bl/6J mice one week prior to biweekly β-glucan treatment and imaged 3 weeks later.ResultsFirst, we found that β-glucan was highly efficient in acutely clearing OvCa cells from the peritoneal fluid. Mechanistically, β-glucan captured free-floating OvCa cells into solid nodules through two non-redundant and equally important pathways: (1) an unexpected Dectin-1/SYK-independent, heparin-sensitive pathway that was mediated by PRMΦ aggregation in the peritoneal fluid; and (2) a Dectin-1/SYK/PAD4-dependent pathway that was mediated by neutrophil extracellular traps in the omentum. Second, we found that β-glucan also completely cleared cancer from the peritoneal fluid and prevented ascites accumulation in advanced disease. Combining β-glucan with IFNγ (β-glucan/IFNγ) not only cleared ascites but also regressed omentum tumors and prevented intraperitoneal metastases as compared to PBS-, β-glucan-, or IFNγ-treated mice.ConclusionsIntraperitoneal injection of β-glucan clears OvCa cells from the peritoneal fluid and has therapeutic potential to control OvCa metastasis.

BackgroundEradication of intraperitoneal metastasis of ovarian cancer (OC) remains an unmet challenge. Current T cell-mediating immunotherapies have not been applied to OC due to lack of efficacy in most patients. Immunosuppressive myeloid cells associate with tumor progression and treatment resistance in the metastatic sites of OC. The purpose of this study is to develop a novel immunotherapy that activates myeloid cells with the goal of eradicating metastatic OC.MethodsRecently established, clinically relevant murine OC cells, that are homologous recombination proficient, KPCA (KRASG12VTrp53R172HCcne1OEAkt2OE ), were intraperitoneally injected to build the murine metastatic OC model. β-glucan and interferon (IFN) γ were used to activate myeloid cells. β-glucan is a yeast cell wall polysaccharide that is currently in clinical trials to treat multiple cancers. It canonically activates myeloid cells through the Dectin-1 pathway and induces infiltration of monocytes and neutrophils. IFNγ activates macrophages to an anti-tumor status. Tumor burden and tumor microenvironment in the ascites and omentum were evaluated using bioluminescence imaging, confocal imaging, flow cytometry, and single-cell RNA sequencing (scRNA seq) to investigate whether and how β-glucan modulates metastatic OC with or without IFNγ. Furthermore, OC patient survival was analyzed based on gene expression using public dataset. Finally, combination of myeloid cell activation with carboplatin was tested.Resultsβ-glucan alone was efficient in clearing ascites, although it did not affect total metastases. On the other hand, combining β-glucan with IFNγ (β-glucan/IFNγ) not only cleared ascites but also reduced total metastases compared to PBS-, β-glucan-, or IFNγ-treated mice. This anti-tumor immunity required T cells and non-tumor IFNγ signaling in the host. scRNA seq of omental tumors revealed β-glucan/IFNγ induced the enrichment of a unique subset of neutrophils and macrophages compared to other groups. The neutrophil subset upregulated Camp and Ltf, which are granule proteins known to have tumoricidal function. Eukaryotic initiation factor-2 signaling, which is associated with reactive oxygen species (ROS) production, is the most upregulated pathway in the neutrophil subset. The macrophage subset selectively expressed interleukin (IL)-27, which has anti-tumor potential mainly through T cells. Blocking IL-27 significantly impaired the anti-tumor response of β-glucan/IFNγ. OC patients with high expression of these genes identified in the novel myeloid cell subsets correlate with better overall survival. Finally, combining β-glucan/IFNγ with carboplatin nearly eradicated chemoresistant KPCA tumors.Conclusionsβ-glucan/IFNγ suppressed metastatic OC enriching anti-tumor myeloid cell populations. Combination therapy of this myeloid cell activation and standard-of-care chemotherapy could potentially transform treatments against metastatic OC.Ethics ApprovalThis study is approved by IACUC (#201536).

Ovarian Cancer (OvCa) is the fifth leading cause of cancer-related death in women and the deadliest gynecologic malignancy. Unfortunately, despite a generally positive response to chemotherapy following surgical debulking., most OvCa patients will relapse—now having developed resistance to chemotherapy. Nearly a decade ago, our lab discovered that the hydrogen sulfide (H2S)—producing enzyme, cystathionine beta synthase (CBS), is significantly upregulated in OvCa and contributes to chemotherapy resistance. Here, we expand on the role of CBS in the progression OvCa by demonstrating its role in maintaining the stability of two proteins, mitofusin 2 (MFN2) and specificity protein 1 (SP1). MFN2 is one of two primary outer mitochondria membrane (OMM) fusion proteins. MFN2 is also present on the surface of the endoplasmic reticulum (ER) and is considered to be critical for the tethering of the ER and the mitochondria to each other. Therefore, MFN2 is necessary to maintain proper mitochondria network fusion, ATP synthesis, and Ca2+ homeostasis -processes which are known to promote cell function and survival. In chapters II and III we show how CBS/H2S antioxidant properties support MFN2 stability in not only OvCa cells themselves, but also another primary cell found in the tumor microenvironment, endothelial cells. SP1 is a ubiquitous transcription factor known to regulate the transcriptional activity of many genes involved in most cellular processes, including the processes of lipogenesis (de novo lipid synthesis). Lipogenesis can support cancer cell growth and survival by providing lipids for the generation of cell membranes, lipids for energy utilization, and lipids for lipid signaling. Here we report that CBS promotes SP1 stability via a H2S-dependent posttranslational modification, persulfidation. Taken together, the data presented in this thesis supports the notion that CBS is a viable therapeutic target for the treatment of OvCa as it not only controls multiple pro-tumorigenic processes in OvCa cells, but may also mediate the tumor microenvironment crosstalk.

Patients with metastatic ovarian cancer (OvCa) have a 5-year survival rate of less than 30% due to persisting dissemination of chemoresistant cells in the peritoneal fluid and the immunosuppressive microenvironment in the peritoneal cavity. Here, we report that intraperitoneal administration of β-glucan and IFNγ (BI) induced robust tumor regression in clinically relevant models of metastatic OvCa. BI induced tumor regression by controlling fluid tumor burden and activating localized antitumor immunity. β-glucan alone cleared ascites and eliminated fluid tumor cells by inducing intraperitoneal clotting in the fluid and Dectin-1-Syk-dependent NETosis in the omentum. In omentum tumors, BI expanded a novel subset of immunostimulatory IL27+ macrophages and neutralizing IL27 impaired BI efficacy . Moreover, BI directly induced IL27 secretion in macrophages where single agent treatment did not. Finally, BI extended mouse survival in a chemoresistant model and significantly improved chemotherapy response in a chemo-sensitive model. In summary, we propose a new therapeutic strategy for the treatment of metastatic OvCa.

High-grade serous carcinoma (HGSC) of the ovary acquires chemoresistance through diverse cancer cell-intrinsic and-extrinsic mechanisms, culminating in treatment-refractory intraperitoneal metastasis. How chemotherapy-induced remodeling of the tumor microenvironment modulates drug sensitivity remains unclear. In this study, we demonstrate that chemotherapy induced IL1β-dependent neutrophil accumulation in tumors, driving chemoresistance in HGSC. Using patient samples, bulk transcriptomic profiling before and after chemotherapy revealed post-treatment upregulation of IL1B, and single-cell RNA sequencing identified myeloid cells as its principal source. In a chemoresistant murine metastatic ovarian cancer model, chemotherapy increased neutrophils and neutrophil extracellular traps (NETs) in omentum tumors; these increases were abrogated in IL1β-deficient mice, with expansion of activated CD8+ T cells and tumor control. Neutrophil depletion in wild-type mice recapitulated the chemosensitive phenotype of IL1β-deficient mice. In vitro, IL1β did not alter cancer cell-intrinsic chemosensitivity, whereas NETs reduced the chemosensitivity of cancer cells. Additionally, the dominant IL1β receptor (IL1R1) was predominantly expressed in tumor-associated fibroblasts in humans and mice. Consistently, IL1R1-deficient mice exhibited chemosensitivity with decreased neutrophil accumulation and increased IFNγ⁺TNF⁺CD8⁺ T cells. We also found that chemotherapy upregulated CXCL2 in patients and that ablating IL1β-IL1R1 axis decreased CXCL2 expression in tumor-associated fibroblasts in mice. Finally, residual human HGSC tumor after chemotherapy showed increased neutrophils and a trend toward more NETs. Collectively, these findings illuminate a paradoxical, cancer cell-extrinsic mechanism in HGSC whereby chemotherapy itself amplifies chemoresistance and suggest that targeting chemotherapy-induced inflammation may help overcome treatment resistance.