title: Adipose-derived stromal cells for tissue engineering after breast cancer surgery
Creators: name: Challapalli, Ritihaas Surya
Publisher: name: University of Galway
Year: 2024
Subjects: subject: Medicine
subject: Surgery
subject: Tissue engineering
subject: Breast cancer surgery

description: Surgical resection is the cornerstone of effective breast cancer management and up to 40% of patients require mastectomy to achieve locoregional disease control. This can result in negative psychosocial and quality of life outcomes. Breast reconstruction following mastectomy and volume replacement following breast conserving surgery have been shown to improve aesthetic and quality-of-life outcomes in breast cancer patients. However, current reconstruction techniques which include implant-based or autologous reconstruction are limited by potential morbidity and suboptimal aesthetic outcomes. There is an urgent requirement for new and improved approaches to breast reconstruction. Adipose tissue engineering is a novel approach where autologous Adipose-derived stromal cells (ADSCs) are combined with a biocompatible scaffold to generate adipose tissue which can be utilised to replace volume lost through surgical resection and presents exciting potential for post-surgical reconstruction in breast cancer patients. However, concerns have been raised about the suitability of ADSCs as a regenerative tool in the oncological setting due to their wound healing and paracrine signalling capabilities and the effects of breast cancer management therapies on ADSCs is poorly understood. Before this approach can be translated to the clinical setting for breast cancer patients, it is imperative that both feasibility and oncologic safety are investigated. This study hypothesizes that combining modified hyaluronic acid (HA) hydrogels with patient-derived adipose-derived stem cells (ADSCs) is a suitable and effective approach for adipose tissue engineering. The aims of the study are to investigate the impact of systemic anti-oestrogen therapy on the function and regenerative capacity of ADSCs from breast cancer patients, evaluate the suitability of modified HA hydrogels combined with ADSCs for adipose tissue engineering in vitro, and assess the safety and efficacy of the ADSC-HA hydrogel combination for tissue regeneration post-tumour resection in a murine model. xv The first part of this thesis investigates the role of systemic anti-oestrogen therapy on the function and regenerative capacity of ADSCs from breast cancer patients to elucidate the applicability of ADSC-based regenerative therapies in combination with breast cancer treatment. We hypothesised that if ADSCs express Oestrogen receptors their function and regenerative capacity may be negatively impacted by systemic anti-oestrogen therapy. To address this ADSCs were exposed to Tamoxifen’s active metabolites - Afimoxifene (4-Hydroxy-Tamoxifen), Endoxifen (N-desmethyl-4-hydroxytamoxifen) and their combination (Tamoxifen). Following this exposure, the function and regenerative capacity of the ADSCs were assessed through various assays including cell viability, apoptosis, adipogenic differentiation, and gene expression analysis. These assays were performed to gain insights into how Tamoxifen and its metabolites may impact the ADSCs in terms of their survival, programmed cell death, ability to differentiate into adipocytes, and alterations in Adipogenic gene expression profiles. We found that ADSCs tolerated doses of up to 1μM drug with no decline in cell viability or increase in caspase 3/7 dependent apoptosis. ADSCs exhibited no functional decline in adipogenic differentiation or gene expression at physiologically relevant doses but had a downward trend in Adipogenic gene expression at supra-physiological doses. VEGF165 protein expression was also not significantly impacted at physiologically relevant doses but demonstrated an insignificant decline at higher drug concentrations. Based on this evidence, we concluded that simultaneous Tamoxifen therapy for breast cancer management will not adversely affect the efficacy of ADSC-based regenerative therapy. The second part of the study investigates the suitability of Hyaluronic acid (HA) derivatives combined with ADSCs for adipose tissue engineering, in-vitro. We hypothesise that the biomechanical properties of modified HA hydrogels would be suitable/supportive of ADSC attachment, proliferation and differentiation. First, the cell seeding density and hydrogel concentration of the ADSC-HA hydrogel mixture were optimized. Subsequently, the optimized xvi ADSC-HA hydrogels were evaluated for their stiffness (Young's modulus), cell metabolic activity, viability, and adipokine secretion profile. The findings revealed that the ADSC-HA hydrogels remained stable and exhibited mechanical stiffness similar to that of breast adipose tissue when infused with ADSCs. Moreover, the encapsulated cells within the hydrogels were found to be viable, exhibited proliferation, and successfully differentiated into mature adipocytes in vitro (3D culture). The final part of the thesis, investigates the safety and efficacy of combining ADSCs and HA-Hydrogel as an approach to tissue regeneration post tumour resection in-vivo (pre-clinical murine model). A murine breast cancer and reconstruction model was established by subcutaneous implantation of breast cancer cells into immunocompetent Balb/C mice. Tumours were then surgically resected and ADSCs ± hydrogels or hydrogels- only were introduced to the tumour cavity as a breast reconstruction. The animals were monitored for tumour recurrence, graft volume and adverse reactions. The animals were euthanized after 4 weeks and tissues were harvested for immunohistochemistry. We found that both the ADSC+hydrogel and the hydrogel-only groups exhibited similar mean weight and volume of excised reconstructed tissues. Additionally, both of these groups showed a significant increase in weight and volume of reconstructed tissue compared to the cells alone group. Immunohistochemistry performed on paraffin-embedded reconstructed tissue (Hydrogel ±ADSC) showed formation of mature adipocytes to the periphery and vascular structures throughout the reconstructed tissue. Capsule formation was observed in both groups (ADSC+ Hydrogel and Hydrogel-only) with insignificant immune cell infiltration or response. These finding indicate that the ADSC-hydrogel supports mature adipocyte formation with minimal to no immune response but may cause capsule formation. Further long term evaluation of the ADSC-hydrogel in murine and large animal models is necessary to fully elucidate the graft integration and survival in-vivo.