Advanced Glycation End Products as a Potential Target for Restructuring the Ovarian Cancer Microenvironment: A Pilot Study

Abstract

Ovarian cancer is the sixth leading cause of cancer-related death in women, and both occurrence and mortality are increased in women over the age of 60. There are documented age-related changes in the ovarian cancer microenvironment that have been shown to create a permissive metastatic niche, including the formation of advanced glycation end products, or AGEs, that form crosslinks between collagen molecules. Small molecules that disrupt AGEs, known as AGE breakers, have been examined in other diseases, but their efficacy in ovarian cancer has not been evaluated. The goal of this pilot study is to target age-related changes in the tumor microenvironment with the long-term aim of improving response to therapy in older patients. Here, we show that AGE breakers have the potential to change the omental collagen structure and modulate the peritoneal immune landscape, suggesting a potential use for AGE breakers in the treatment of ovarian cancer.

Keywords: AGE breakers; ALT-711; Alagebrium; advanced glycation end product (AGE); collagen; microenvironment; omentum; ovarian cancer.

Figure 1

Presence of advanced glycation end products (AGEs) in the omental microenvironment. (a) Omenta (n = 7/group) harvested from young (3–6 month) or aged (20–23 month) mice were stained with a fluorescently tagged α-AGE antibody and imaged with a multiphoton microscope in the SHG (collagen, grey) and fluorescent (AGE, green) channels, showing the presence of AGEs in the omental microenvironment. Scale bar 100 μm. (b) Quantification of AGE staining showing the average AGE staining intensity per mouse (p = 0.0130) and the area stained by the fluorescent AGE antibody (p = 0.6430). (* p < 0.05).

Figure 2

Experimental design. (a) Female mice (young: 3–6 month or aged: 20–23 month) in the control group were given an I.P. injection 5 days/week for 4 weeks of the vehicle (0.8 µm DMSO in PBS). The treatment groups (aged: 20–23 months) were given 1 mg/kg of either ALT-711 or 2C8 in 0.8 µM DMSO in PBS injected I.P. 5 days/week for four weeks. Image made with BioRender. (b) Chemical structures of AGE breakers ALT-711 (also known as Alagebrium) and 2C8. ALT-711 was chosen as it is the industry standard for AGE breakers and has been shown to be safe and effective in FDA clinical trials, and has been shown to have some anti-cancer effects in breast cancer models [22,24]. 2C8 was chosen as it has been shown to have increased AGE-breaking capability as compared to ALT-711 in vitro, but has not been evaluated in vivo [25,26].

Figure 3

Analysis of AGE breaker-treated omental collagen and structure. (a) Omenta (n = 6–7/group) were imaged ex vivo using second harmonic generation (SHG) imaging. Scale bar 100 μm. (b) Omenta (n = 6–7/group) were fixed in 2% OsO₄ and dehydrated, mounted on stubs with carbon stickers, silver painted, and coated with iridium, then imaged using scanning electron microscopy (SEM). Scale bar 500 μm.

Figure 4

OvCa cell adhesion. (a) Omental explants (n = 3/group) were incubated with 10⁶ RFP-tagged ID8^Trp53-/- cells for 2 h prior to gentle washing and imaging. Scale bars denote 300 μm (4×) and 100 μm (10×) (b) RFP signal was quantified to analyze adhesion, shown as total area of RFP signal. Average cell area and average cell compactness were measured using Cell Profiler. p values listed in Table A1 in Appendix A.

Figure 5

Analysis of peritoneal lymphocyte immune populations. Mice (n = 3/group) were sacrificed, and peritoneal immune cells were harvested via peritoneal lavage and stained for multiplex flow cytometry. Immune types stained for included (a) total lymphocytes, (b) B cells, (c) total T cells, (d) CD4+ T cells, (e) regulatory T cells (Tregs), (f) CD8+ T cells, and (g) TCRγδ+ T cells. Student’s t-test determined significance (* p < 0.05 and *** p < 0.0005). p values listed in Table A1 in Appendix A. Antibodies are listed in Table A2 and Table A3 in Appendix A.

Figure 6

Analysis of peritoneal non-lymphocyte immune populations. Mice (n = 3/group) were sacrificed, and peritoneal immune cells were harvested via peritoneal lavage and stained for multiplex flow cytometry. Immune types stained for included (a) granulocytes, (b) monocytes, (c) natural killer (NK) cells, (d) macrophages, (e) M2 macrophages, and (f) Tim4+ tissue-resident macrophages. Student’s t-test determined significance (* p < 0.05). p values listed in Table A1 in Appendix A. Antibodies are listed in Table A2 and Table A3 in Appendix A.