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

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.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

Development of a Novel Humanized Single Chain Antibody-Streptococcal Superantigen-Derived Immunotherapy Targeting the 5T4 Oncofetal Antigen

Superantigens (SAgs) are microbial toxins that cross-link T cell receptors with major histocompatibility complex (MHC) class II (MHC II) molecules leading to the activation of large numbers of T cells. Herein, the development and preclinical testing of a novel tumour-targeted SAg (TTS) therapeutic built using the streptococcal pyrogenic exotoxin C (SpeC) SAg and targeting cancer cells expressing the 5T4 tumour-associated antigen (TAA) was described. To inhibit potentially harmful widespread immune cell activation, a SpeC mutation within the high-affinity MHC II binding interface was generated (SpeCD203A) that demonstrated a pronounced reduction in mitogenic activity, yet this mutant could still induce immune cell-mediated cancer cell death in vitro. To target 5T4+cancer cells, a humanized single-chain variable fragment (scFv) antibody to recognize 5T4 (scFv5T4) was engineered. Specific targeting of scFv5T4 was verified. SpeCD203A used to scFv5T4 maintained the ability to activate and induce immune cell-mediated cytotoxicity of colon cancer cells. Using a xenograft model of established human colon cancer, it was demonstrated that the SpeC-based TTS was able to control the growth and spread of large tumours in vivo. This required both TAA targeting by scFv5T4 and functional SAg activity. These studies lay the foundation for the development of streptococcal SAgs as \u27next-generation\u27 TTSs for cancer immunotherapy

FOLFIRINOX chemotherapy modulates the peripheral immune landscape in pancreatic cancer:Implications for combination therapies and early response prediction

Background: FOLFIRINOX chemotherapy has improved outcomes for pancreatic cancer patients, but poor long-term survival outcomes and high toxicity remain challenges. This study investigates the impact of FOLFIRINOX on plasma proteins and peripheral immune cells to guide immune-based combination therapies and, ideally, to identify a potential biomarker to predict early disease progression during FOLFIRINOX. Methods: Blood samples were collected from 86 pancreatic cancer patients before and two weeks after the first FOLFIRINOX cycle and subjected to comprehensive immune cell and proteome profiling. Principal Component Analysis and Linear Mixed Effect Regression models were used for data analysis. FOLFIRINOX efficacy was radiologically evaluated after the fourth cycle. Results: One cycle of FOLFIRINOX diminished tumour-cell-related pathways and enhanced pathways related to immune activation, illustrated by an increase in pro-inflammatory IL–18, IL–15, and TNFRSF4. Similarly, FOLFIRINOX promoted the activation of CD4 + and CD8 + T cells, the proliferation of NK(T), and the activation of antigen-presenting cells. Furthermore, high pre-treatment levels of VEGFA and PRDX3 and an elevation in FCRL3 levels after one cycle predicted early progression under FOLFIRINOX. Finally, patients with progressive disease exhibited high levels of inhibitory markers on B cells and CD8 + T cells, while responding patients exhibited high levels of activation markers on CD4 + and CD8 + T cell subsets. Conclusion: FOLFIRINOX has immunomodulatory effects, providing a foundation for clinical trials exploring immune-based combination therapies that harness the immune system to treat pancreatic cancer. In addition, several plasma proteins hold potential as circulating predictive biomarkers for early prediction of FOLFIRINOX response in patients with pancreatic cancer.</p

Digital image analysis: Predictive biomarkers for chemoimmunotherapy in malignant pleural mesothelioma

No abstract availabl

Neoadjuvant immunotherapy in primary and metastatic colorectal cancer

Background Colorectal cancer (CRC) is the second most common solid organ cancer. Traditional treatment is with surgery and chemotherapy. Immunotherapy has recently emerged as a neoadjuvant therapy that could change treatment strategy in both primary resectable and metastatic CRC. Methods A literature review of PubMed with a focus on studies exploring upfront immunotherapy in operable CRC, either for primary resectable stage I–III cancers or for (potentially) operable liver metastasis. Results Immune checkpoint blockade by the programmed cell death 1 (PD-1) receptor inhibitors nivolumab and pembrolizumab and the cytotoxic T cell-associated protein 4 (CTLA-4) inhibitor ipilimumab has shown good results in both early-stage and advanced CRC. The effects of immune checkpoint inhibitors have so far been demonstrated in small phase I/II studies and predominantly in treatment-refractory stage IV disease with defect Mismatch repair (dMMR). However, recent data from phase I/II (NICHE-1) studies suggest an upfront role for immunotherapy in operable stage I–III disease. By blocking crucial immune checkpoints, cytotoxic T cells are activated and release cytotoxic signals that initiate cancer cell destruction. The very high complete response rate in dMMR operable CRC with neoadjuvant immunotherapy with nivolumab and ipilimumab, and even partial pathological response in some patients with proficient MMR (pMMR) CRC, calls for further attention to patient selection for neoadjuvant treatment, beyond MMR status alone. Conclusion Early data on the effect of immunotherapy in CRC provide new strategic thinking of treatment options in CRC for both early-stage and advanced disease, with prospects for new trials.publishedVersio

Interactions between anti-EGFR therapies and cytotoxic chemotherapy in oesophageal squamous cell carcinoma: why clinical trials might have failed and how they could succeed

Acknowledgements We thank Alice Savage for technical laboratory assistance. Funding The work undertaken was funded by Ninewells Cancer Campaign (Dundee) and Scottish Government Chief Scientist Office (Grant reference TCS/19/18).Peer reviewedPublisher PD