Intracellular pH (pHi) dynamics are critical for regulating normal cell physiology. For example, transient increases in pHi (7.2−7.6) regulate cell behaviors like cell polarization, actin cytoskeleton remodeling, and cell migration. Most studies on pHdependent cell behaviors have been performed at the population level and use nonspecific methods to manipulate pHi. The lack of tools to specifically manipulate pHi at the single-cell level has hindered investigation of the role of pHi dynamics in driving single cell behaviors. In this work, we show that Archaerhodopsin (ArchT), a light-driven outward proton pump, can be used to elicit robust and physiological pHi increases over the minutes time scale. We show that activation of ArchT is repeatable, enabling the maintenance of high pHi in single cells for up to 45 minutes. We apply this spatiotemporal pHi manipulation tool to determine whether increased pHi is a sufficient driver of membrane ruffling in single cells. Using the ArchT tool, we show that increased pHi in single cells can drive localized membrane ruffling responses within seconds and increased membrane dynamics (both protrusion and retraction events) compared to unstimulated ArchT cells as well as control cells. Overall, this tool allows us to directly investigate the relationship between increased pHi and single cell behaviors such as membrane ruffling. This tool will be transformative in facilitating experiments that are required to determine roles for increased pHi in driving single cell behaviors.

Background: Age is the most significant risk factor for ovarian cancer (OvCa), the deadliest gynecologic malignancy. Metastasizing OvCa cells adhere to the omentum, a peritoneal structure rich in collagen, adipocytes, and immune cells. Ultrastructural changes in the omentum and the omental collagen matrix with aging have not been evaluated.

Intracellular pH (pHi) dynamics are critical for regulating normal cell physiology. For example, transient increases in pHi (7.2-7.6) regulate cell behaviors like cell polarization, actin cytoskeleton remodeling, and cell migration. Most studies on pH-dependent cell behaviors have been performed at the population level and use non-specific methods to manipulate pHi. The lack of tools to specifically manipulate pHi at the single-cell level has hindered investigation of the role of pHi dynamics in driving single cell behaviors. In this work, we show that Archaerhodopsin (ArchT), a light-driven outward proton pump, can be used to elicit robust and physiological pHi increases over the minutes timescale. We show that activation of ArchT is repeatable, enabling the maintenance of high pHi in single cells for approximately 45 minutes. We apply this spatiotemporal pHi manipulation tool to determine whether increased pHi is a sufficient driver of membrane ruffling in single cells. Using the ArchT tool, we show that increased pHi in single cells can drive localized membrane ruffling responses within seconds and 2 increased membrane dynamics (both protrusion and retraction events) compared to control cells. Overall, this tool allows us to directly investigate the relationship between increased pHi and cell behaviors such as membrane ruffling. This tool will be transformative in facilitating the experiments required to determine if increased pHi is a driver of these cell behaviors at the single-cell level.

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. Using a C57Bl/6 mouse model of aging, young (Y) mice ranging from 3-6 months of age and aged (A) mice ranging from 20-23 months of age were used to study the role aging has on metastasis. Fluorescently tagged C57Bl/6 syngeneic ID8trp53-/- mouse ovarian surface epithelial cancer cells were injected intraperitoneally in Y and A mice and disease progression was evaluated following 5.5 weeks. Organ-specific tumor burden was quantified with ImageJ, revealing increased tumor burden in A mice compared to their Y counterparts. These results were reproduced in the FVB mouse model using syngeneic PTENshRNA/KRASG12V oviductal epithelial cells. While in vitro assays showed no significant difference in adhesion or invasion of OvCa cells on Y vs A collagen, human cell lines 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, second harmonic generation microscopy (SHG) and CHP fluorescence analysis of early tumor events shows increased peritumoral collagen remodeling in Y 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. To further investigate peritumoral collagen in the metastatic microenvironment, 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 with scanning electron microscopy (SEM). In conclusion, aging induces changes in the structure and properties of both intra- and peritumoral collagen, increasing OvCa metastatic success. Citation Format: Elizabeth Harper, Elizabeth Agadi, Emma Sheedy, Preston Carey, Paul Wilkinson, Michael Siroky, Tyvette Hilliard, Ethan Low, Annemarie Leonard, Yueying Liu, Jing Yang, M. Sharon Stack. Age-related changes in microenvironmental collagen affect ovarian cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3641.