Endocytic recycling regulates extracellular matrix assembly by cancer associated fibroblasts to promote ovarian cancer cell invasion

Abstract

High-grade serous ovarian cancer (HGSOC) is the most common and lethal form of ovarian cancer. Early-stage diagnosis is rare due to the subtle nature of symptoms, and at later stages metastasis is common. The omentum is a preferential site for metastatic tumour formation, as it provides a HGSOC supportive tumour microenvironment (TME), an aspect of which is the extracellular matrix (ECM), assembled primarily by cancer associated fibroblasts (CAFs). Whilst it is known that fibrillar collagen-rich ECM in the omentum is associated with HGSOC progression, the molecular mechanisms involved in ECM assembly within the TME are poorly understood. Although endocytic recycling has been implicated in basement membrane ECM assembly, and in the remodelling of the ECM protein fibronectin, its role in ECM assembly within the HGSOC TME remains unclear. We found that Kuramochi HGSOC cells were minimally invasive in collagen hydrogels, but their co-culture with CAFs isolated from HGSOC metastases within spheroids significantly enhanced invasion. CAF-conditioned medium was insufficient to promote HGSOC invasion, however substantial collagen remodelling was detected in the vicinity of co-culture spheroids, suggesting that CAFs directly influenced the ECM to support invasion. An siRNA screening approach was used to deplete key endocytic recycling regulator Rab GTPases, and Arf6, in CAFs to identify those required to promote HGSOC invasion. Depletion of Arf6, or Rab4a and Rab4b in combination, resulted in a reduced capacity of two primary CAF lines to facilitate HGSOC invasion in 3D. We next analysed the influence of Arf6, Rab14, Rab4a and Rab4b, on ECM assembly. Fibronectin is thought to be important in generating fibrillar collagen matrix, however its assembly was unaffected by recycling regulator depletion. Instead, depletion of Arf6, or Rab14, inhibited collagen I deposition by CAFs, with collagen monomer secretion remaining unaffected. Recently, the uptake of secreted collagen and it’s recycling via an unknown route, has been implicated in fibrillar collagen assembly. Measuring the uptake of exogenous collagen I monomers by CAFs, demonstrated that Arf6 depletion significantly elevated collagen I uptake. However, there was a significant delay in the reappearance of internalised collagen as fibrils at the cell surface, suggesting that Arf6 regulates the deposition of endocytosed and recycled collagen. Furthermore, live-cell imaging revealed that Arf6 localised to sites of exogenous collagen fibrillogenesis in CAFs overexpressing Arf6-mNeonGreen. Together, our data demonstrate key roles for endocytic recycling in the support of HGSOC progression by CAFs, and specifically implicate Arf6 in collagen assembly within the TME.

Date of Award	31 Dec 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Patrick Caswell (Supervisor) & Richard Edmondson (Supervisor)