Instructor, Obstetrics & Gynecology - Gynecologic Oncology
Densely O-glycosylated mucin domains are found in a broad range of cell surface and secreted proteins, where they play key physiological roles. In addition, alterations in mucin expression and glycosylation are common in a variety of human diseases, such as cancer, cystic fibrosis, and inflammatory bowel diseases. These correlations have been challenging to uncover and establish because tools that specifically probe mucin domains are lacking. Here, we present a panel of bacterial proteases that cleave mucin domains via distinct peptide- and glycan-based motifs, generating a diverse enzymatic toolkit for mucin-selective proteolysis. By mutating catalytic residues of two such enzymes, we engineered mucin-selective binding agents with retained glycoform preferences. StcEE447D is a pan-mucin stain derived from enterohemorrhagic Escherichia coli that is tolerant to a wide range of glycoforms. BT4244E575A derived from Bacteroides thetaiotaomicron is selective for truncated, asialylated core 1 structures commonly associated with malignant and premalignant tissues. We demonstrated that these catalytically inactive point mutants enable robust detection and visualization of mucin-domain glycoproteins by flow cytometry, Western blot, and immunohistochemistry. Application of our enzymatic toolkit to ascites fluid and tissue slices from patients with ovarian cancer facilitated characterization of patients based on differences in mucin cleavage and expression patterns.
View details for DOI 10.1073/pnas.2012196117
View details for PubMedID 32817557
View details for Web of Science ID 000497337700099
View details for Web of Science ID 000497337700104
View details for Web of Science ID 000525055501242
Ovarian cancer and triple-negative breast cancer are among the most lethal diseases affecting women, with few targeted therapies and high rates of metastasis. Cancer cells are capable of evading clearance by macrophages through the overexpression of anti-phagocytic surface proteins called 'don't eat me' signals-including CD471, programmed cell death ligand 1 (PD-L1)2 and the beta-2 microglobulin subunit of the major histocompatibility class I complex (B2M)3. Monoclonal antibodies that antagonize the interaction of 'don't eat me' signals with their macrophage-expressed receptors have demonstrated therapeutic potential in several cancers4,5. However, variability in the magnitude and durability of the response to these agents has suggested the presence of additional, as yet unknown 'don't eat me' signals. Here we show that CD24 can be the dominant innate immune checkpoint in ovarian cancer and breast cancer, and is a promising target for cancer immunotherapy. We demonstrate a role for tumour-expressed CD24 in promoting immune evasion through its interaction with the inhibitory receptor sialic-acid-binding Ig-like lectin 10 (Siglec-10), which is expressed by tumour-associated macrophages. We find that many tumours overexpress CD24 and that tumour-associated macrophages express high levels of Siglec-10. Genetic ablation of either CD24 or Siglec-10, as well as blockade of the CD24-Siglec-10 interaction using monoclonal antibodies, robustly augment the phagocytosis of all CD24-expressing human tumours that we tested. Genetic ablation and therapeutic blockade of CD24 resulted in a macrophage-dependent reduction of tumour growth in vivo and an increase in survival time. These data reveal CD24 as a highly expressed, anti-phagocytic signal in several cancers and demonstrate the therapeutic potential for CD24 blockade in cancer immunotherapy.
View details for DOI 10.1038/s41586-019-1456-0
View details for PubMedID 31367043
The mammalian glycocalyx is a heavily glycosylated extramembrane compartment found on nearly every cell. Despite its relevance in both health and disease, studies of the glycocalyx remain hampered by a paucity of methods to spatially classify its components. We combine metabolic labeling, bioorthogonal chemistry, and super-resolution localization microscopy to image two constituents of cell-surface glycans, N-acetylgalactosamine (GalNAc) and sialic acid, with 10-20 nm precision in 2D and 3D. This approach enables two measurements: glycocalyx height and the distribution of individual sugars distal from the membrane. These measurements show that the glycocalyx exhibits nanoscale organization on both cell lines and primary human tumor cells. Additionally, we observe enhanced glycocalyx height in response to epithelial-to-mesenchymal transition and to oncogenic KRAS activation. In the latter case, we trace increased height to an effector gene, GALNT7. These data highlight the power of advanced imaging methods to provide molecular and functional insights into glycocalyx biology.
View details for DOI 10.1016/j.devcel.2019.04.035
View details for PubMedID 31105009
The complex tumor microenvironment in gynecologic cancers plays a major role in modulating anti-tumor immune responses. The interaction of cancer cells with the diverse spectrum of immune effector cells has an important impact on the efficacy of standard chemotherapy and novel immunotherapy approaches. In this review, we specifically focus on the role of macrophages in ovarian, endometrial and cervical cancers. We discuss the origins of macrophages and their polarization state dictated by the microenvironment's cues. Within the tumor niche, tumor-associated macrophages (TAMs) promote tumor growth and mediate immune-suppression thereby effecting treatment responses. We outline clinical strategies that directly target TAMs, including inhibition of macrophage differentiation, prevention of the recruitment of monocytes to the tumor, enhancement of phagocytosis and immune checkpoint blockade.
View details for PubMedID 29395307
To provide an overview of the principles, safety and efficacy of adoptive cell therapy (ACT) in solid tumors particularly in gynecological cancers.Efforts to target solid tumors using tumor-infiltrating lymphocytes and genetically modified T cells have shown promising efficacy in some patients. Two food and drug administration approvals for the treatment of leukemia are the first gene therapies available for cancer treatment in the United States.Genetic engineering of antitumor immunity using T cells has the potential to target specific tumor-associated antigens and overcome obstacles to successful immunotherapy like immune-suppressive factors in the tumor microenvironment.
View details for PubMedID 29227304
Tumor-associated macrophages (TAMs) are often associated with a poor prognosis in cancer. To gain a better understanding of cellular recruitment and dynamics of TAM biology during cancer progression, we established a novel transgenic mouse model for in vivo imaging of luciferase-expressing macrophages.B6.129P2-Lyz2(tm1(cre)Ifo)/J mice, which express Cre recombinase under the control of the lysozyme M promoter (LysM) were crossed to Cre-lox Luc reporter mice (RLG), to produce LysM-LG mice whose macrophages express luciferase. Cell-type-specific luciferase expression in these mice was verified by flow cytometry, and via in vivo bioluminescence imaging under conditions where macrophages were either stimulated with lipopolysaccharide or depleted with clodronate liposomes. The distribution of activated macrophages was longitudinally imaged in two immunocompetent LysM-LG mouse models with either B16 melanoma or ID8 ovarian cancer cells.In vivo imaging of LysM-LG mice showed luciferase activity was generated by macrophages. Clodronate liposome-mediated depletion of macrophages lowered overall bioluminescence while lipopolysaccharide injection increased macrophage bioluminescence in both the B16 and ID8 models. Tracking macrophages weekly in tumor-bearing animals after intraperitoneal (i.p.) or intraovarian (i.o.) injection resulted in distinct, dynamic patterns of macrophage activity. Animals with metastatic ovarian cancer after i.p. injection exhibited significantly higher peritoneal macrophage activity compared to animals after i.o. injection.The LysM-LG model allows tracking of macrophage recruitment and activation during disease initiation and progression in a noninvasive manner. This model provides a tool to visualize and monitor the benefit of pharmacological interventions targeting macrophages in preclinical models.
View details for DOI 10.1007/s11307-017-1061-2
View details for PubMedID 28233218
Immunotherapy aims to develop combination approaches that simultaneously augment immunity while preventing local immune suppression. Despite advances in combinatorial chemotherapy regimens and the advent of intraperitoneal chemotherapy administration, current therapeutic options for patients with ovarian cancer are inadequate. Advances in immunotherapy offer a promising frontier for treating ovarian tumors. Multiple immunotherapeutic modalities are currently developed and tested in clinical trials. Antibody-based therapies, immune checkpoint blockade, cancer vaccines, and chimeric antigen receptor-modified T cells have demonstrated preclinical success and entered clinical testing. In this review, we discuss these promising immunotherapeutic approaches and emphasize the importance of combinatorial treatment strategies and biomarker discovery.
View details for DOI 10.1016/j.currproblcancer.2016.11.003
View details for Web of Science ID 000398869800004
High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.
View details for DOI 10.3791/52721
View details for PubMedID 26555178
Host tissue microenvironment plays key roles in cancer progression and colonization of secondary organs. One example is ovarian cancer, which colonizes the peritoneal cavity and especially the omentum. Our research indicates that the interaction of ovarian cancer cells with the omental microenvironment can activate a stress-kinase pathway involving the mitogen-activated protein kinase kinase 4 (MKK4). A combination of clinical correlative and functional data suggests that MKK4 activation suppresses growth of ovarian cancer cells lodged in omentum. These findings prompted us to turn our focus to the cellular composition of the omental microenvironment and its role in regulating cancer growth. In this review, in addition to providing an overview of MKK4 function, we highlight a use for metastasis suppressors as a molecular tool to study cancer cell interaction with its microenvironment. We review features of the omentum that makes it a favorable microenvironment for metastatic colonization. In conclusion, a broader, evolutionary biology perspective is presented which we believe needs to be considered when studying the evolution of cancer cells within a defined microenvironment. Taken together, this approach can direct new multi-dimensional lines of research aimed at a mechanistic understanding of host tissue microenvironment, which could be used to realize novel targets for future research.
View details for DOI 10.1007/s10555-012-9371-y
View details for Web of Science ID 000309863800017
View details for PubMedID 22706843