Integrative Analysis of the Aging Peritoneum in Metastatic Receptivity

Aging is the biggest risk factor for the development of ovarian cancer (OvCa), the deadliest cancer of the female reproductive system. Despite this, age is understudied in the OvCa field. OvCa cells preferentially metastasize to the omentum, an organ rich in collagen. While many studies have been done on aged collagen in the skin, the effect of aging on omental collagen has not been investigated. A C57Bl/6 mouse model was used to study aging, where young (Y) mice ranging from 3-6 months of age correspond to women 20-30 years of age and aged (A) mice ranging from 20-23 months of age correspond to women 60-67 years of age. Collagen isolated from Y and A mice was shown to have an increase in advanced glycation end products (AGEs) and reduced degradation by matrix metalloproteinases (MMPs). Tail tendons incubated with MMP-1 showed differences in degradation as shown by scanning electron microscopy (SEM). To further investigate age-related changes in collagen in the metastatic microenvironment, second harmonic generation microscopy (SHG) was used to visualize collagen of common metastatic sites from Y and A tumor naïve C57Bl/6 mice. Distinct structural differences were shown in omental collagen in the Y vs A cohorts and validated at the ultrastructural level with SEM. While in vitro assays showed no significant difference in adhesion or proliferation of OvCa cells on Y vs A collagen, human cell line OVCAR5 showed increased invasion through Boyden invasion chambers lined with A collagen compared to Y. The relationship between tumors and aged collagen was further investigated by analysis of collagen hybridizing peptide (CHP) staining of paraffin-embedded omental tumor sections, which shows an increase of intratumoral collagen remodeling in A tumors despite no significant difference in overall collagen amount as shown by trichrome analysis of serial sections. However, SHG and CHP fluorescence analysis of early tumor events shows decreased peritumoral collagen remodeling in A mice, showing a difference between intratumoral collagen and microenvironmental collagen. Immunohistochemical analysis of paraffin-embedded tumor sections showed similar amounts of advanced glycation end products (AGEs) and cancer-associated fibroblasts (CAFs) in Y and A tumors, suggesting intratumoral collagen is newly synthesized by CAFs whereas the collagen in the microenvironment is truly aged. Additionally, the peritoneal immune landscape was investigated using Northern Lights Flow Cytometry immune panels. Changes were identified that can be tumor permissive, with decreases in natural killer (NK) cells, T cells, and NK T cells and increases in M2 macrophages; or tumor suppressive, with increases in total lymphocytes and Tim4+ macrophages. There were also increases in B cells and total macrophages and decreases in granulocytes, which can have mixed effects on tumors. In conclusion, aging induces many changes in the microenvironment that make the aged omentum a permissive metastatic niche for OvCa.