Thesis

 

3D tumour-stroma microfluidic cultures for the assessment of anti-cancer therapies

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Awarding institution
  • University of Strathclyde
Date of award
  • 2022
Thesis identifier
  • T16209
Person Identifier (Local)
  • 201859514
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Abstract
  • Cancer is a highly complex disease composed of a heterogeneous range of cell types within the tumour microenvironment (TME). Despite advances in cancer treatment, there exists a lack of pre-clinical screening systems that represent the true complexity of in vivo tumours. The solid TME plays a crucial role in tumour development and therapy resistance. Established analytical in vitro methods are often too simplistic in their depiction of solid tumours and are primarily based on 2D cultures of immortalised cancer cell lines. Current preclinical assays commonly lack features of the TME and fail to represent the plethora of cell types present in native human tumours. There exists a need for the development of preclinical platforms that provide greater levels of physiological relevance and predictive value to rapidly determine the efficacy of novel anti-tumour agents and their consequential effects on the various cell types present in the TME. Furthermore, personalised in vitro models could be used for assessing patient tissue to increase accuracy of the predictions of treatment outcomes for patients. Immunotherapy is a promising form of cancer treatment that has not yet been widely harnessed towards the treatment of solid tumours and requires improved methods of in vitro assessment. Microfluidic technologies can provide a cost-effective solution through the advantages of miniaturisation where much smaller volumes of reagents and cell numbers are required in comparison to traditional in vitro assays. Many microfluidic models have been developed featuring tumour spheroids and vascular network structures to study tumour angiogenesis and to assess the performance of anti-cancer agents targeting tumour cells and tumour vasculature. Microfluidic assays have also been established for the study of immunotherapies targeting liquid tumours. However, there is a gap in the development of equivalent models for assessing the efficacy of immunotherapeutics targeting solid tumours. Therefore, elements of the TME were identified to integrate into and increase the complexity of current in vitro models and microfluidic technology utilised to achieve the development of novel microfluidic protocols for miniaturized assays that could be utilized for personalised immunotherapy applications. The aims of this work included achieving the assessment of both the cytotoxicity and target specificity of CAR-T cells in 3D TME relevant models and the validation of the in vitro assessment of CAR-T therapy in combination with chemotherapy and checkpoint blockade. Proof-of-concept applications of assays and protocols for nanoparticle drug delivery, tumour stroma interaction and immune-oncology were demonstrated. Specifically, a viable solid tumour-stromal microenvironment was established using a primary breast cancer cell line and characterisation of co-cultures performed via time-lapse imaging and quantification of fluorescence and protein expression. Adaptable protocols were validated and have potential for use in the analysis of various types of immunotherapy with the potential for incorporation of various cancer and TME associated cell types. This thesis also contains the first report of microfluidic technology combined with SERS to assess targeted nanoparticle binding to and penetration of 3D tumour spheroids. In addition, novel ACT methodology and data analysis protocols were developed to present the first report of the assessment of EGFR specific CAR-T cell cytotoxicity and target specificity in a 3D solid tumour-stromal microfluidic model as a monotherapy and in combination with carboplatin chemotherapy and anti-PD-L1 treatment. These miniaturized proof-of-concept systems using small cell numbers and volumes are highly suited for the analysis of patient biopsy tissue and for determining the efficacy of expensive immunotherapy agents to obtain the maximum data output possible. These assays, due to their sample-saving properties, are amenable for precision medicine applications using patient biopsy tissue, as well as providing a general platform for studying TME interactions. Preliminary assays using primary murine gamma delta T cells demonstrated the potential for human biopsy tissue to be used in microfluidic studies for assessing immunotherapy efficacy and present possible future applications in ACT therapy development.
Advisor / supervisor
  • Zagnoni, Michele
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