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

Detection of Estrogen Receptor Alpha and Assessment of Fulvestrant Activity in MCF-7 Tumor Spheroids Using Microfluidics and SERS

Breast cancer is one of the leading causes of cancer death in women. Novel in vitro tools that integrate three-dimensional (3D) tumor models with highly sensitive chemical reporters can provide useful information to aid biological characterization of cancer phenotype and understanding of drug activity. The combination of surface-enhanced Raman scattering (SERS) techniques with microfluidic technologies offers new opportunities for highly selective, specific, and multiplexed nanoparticle-based assays. Here, we explored the use of functionalized nanoparticles for the detection of estrogen receptor alpha (ERα) expression in a 3D tumor model, using the ERα-positive human breast cancer cell line MCF-7. This approach was used to compare targeted versus nontargeted nanoparticle interactions with the tumor model to better understand whether targeted nanotags are required to efficiently target ERα. Mixtures of targeted anti-ERα antibody-functionalized nanotags (ERα-AuNPs) and nontargeted (against ERα) anti-human epidermal growth factor receptor 2 (HER2) antibody-functionalized nanotags (HER2-AuNPs), with different Raman reporters with a similar SERS signal intensity, were incubated with MCF-7 spheroids in microfluidic devices and spectroscopically analyzed using SERS. MCF-7 cells express high levels of ERα and no detectable levels of HER2. 2D and 3D SERS measurements confirmed the strong targeting effect of ERα-AuNP nanotags to the MCF-7 spheroids in contrast to HER2-AuNPs (63% signal reduction). Moreover, 3D SERS measurements confirmed the differentiation between the targeted and the nontargeted nanotags. Finally, we demonstrated how nanotag uptake by MCF-7 spheroids was affected by the drug fulvestrant, the first-in-class approved selective estrogen receptor degrader (SERD). These results illustrate the potential of using SERS and microfluidics as a powerful in vitro platform for the characterization of 3D tumor models and the investigation of SERD activity

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity.

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant

Hospital admission and emergency care attendance risk for SARS-CoV-2 delta (B.1.617.2) compared with alpha (B.1.1.7) variants of concern: a cohort study

Background: The SARS-CoV-2 delta (B.1.617.2) variant was first detected in England in March, 2021. It has since rapidly become the predominant lineage, owing to high transmissibility. It is suspected that the delta variant is associated with more severe disease than the previously dominant alpha (B.1.1.7) variant. We aimed to characterise the severity of the delta variant compared with the alpha variant by determining the relative risk of hospital attendance outcomes. Methods: This cohort study was done among all patients with COVID-19 in England between March 29 and May 23, 2021, who were identified as being infected with either the alpha or delta SARS-CoV-2 variant through whole-genome sequencing. Individual-level data on these patients were linked to routine health-care datasets on vaccination, emergency care attendance, hospital admission, and mortality (data from Public Health England's Second Generation Surveillance System and COVID-19-associated deaths dataset; the National Immunisation Management System; and NHS Digital Secondary Uses Services and Emergency Care Data Set). The risk for hospital admission and emergency care attendance were compared between patients with sequencing-confirmed delta and alpha variants for the whole cohort and by vaccination status subgroups. Stratified Cox regression was used to adjust for age, sex, ethnicity, deprivation, recent international travel, area of residence, calendar week, and vaccination status. Findings: Individual-level data on 43 338 COVID-19-positive patients (8682 with the delta variant, 34 656 with the alpha variant; median age 31 years [IQR 17–43]) were included in our analysis. 196 (2·3%) patients with the delta variant versus 764 (2·2%) patients with the alpha variant were admitted to hospital within 14 days after the specimen was taken (adjusted hazard ratio [HR] 2·26 [95% CI 1·32–3·89]). 498 (5·7%) patients with the delta variant versus 1448 (4·2%) patients with the alpha variant were admitted to hospital or attended emergency care within 14 days (adjusted HR 1·45 [1·08–1·95]). Most patients were unvaccinated (32 078 [74·0%] across both groups). The HRs for vaccinated patients with the delta variant versus the alpha variant (adjusted HR for hospital admission 1·94 [95% CI 0·47–8·05] and for hospital admission or emergency care attendance 1·58 [0·69–3·61]) were similar to the HRs for unvaccinated patients (2·32 [1·29–4·16] and 1·43 [1·04–1·97]; p=0·82 for both) but the precision for the vaccinated subgroup was low. Interpretation: This large national study found a higher hospital admission or emergency care attendance risk for patients with COVID-19 infected with the delta variant compared with the alpha variant. Results suggest that outbreaks of the delta variant in unvaccinated populations might lead to a greater burden on health-care services than the alpha variant. Funding: Medical Research Council; UK Research and Innovation; Department of Health and Social Care; and National Institute for Health Research

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant

Changes in symptomatology, reinfection, and transmissibility associated with the SARS-CoV-2 variant B.1.1.7: an ecological study

Background The SARS-CoV-2 variant B.1.1.7 was first identified in December, 2020, in England. We aimed to investigate whether increases in the proportion of infections with this variant are associated with differences in symptoms or disease course, reinfection rates, or transmissibility. Methods We did an ecological study to examine the association between the regional proportion of infections with the SARS-CoV-2 B.1.1.7 variant and reported symptoms, disease course, rates of reinfection, and transmissibility. Data on types and duration of symptoms were obtained from longitudinal reports from users of the COVID Symptom Study app who reported a positive test for COVID-19 between Sept 28 and Dec 27, 2020 (during which the prevalence of B.1.1.7 increased most notably in parts of the UK). From this dataset, we also estimated the frequency of possible reinfection, defined as the presence of two reported positive tests separated by more than 90 days with a period of reporting no symptoms for more than 7 days before the second positive test. The proportion of SARS-CoV-2 infections with the B.1.1.7 variant across the UK was estimated with use of genomic data from the COVID-19 Genomics UK Consortium and data from Public Health England on spike-gene target failure (a non-specific indicator of the B.1.1.7 variant) in community cases in England. We used linear regression to examine the association between reported symptoms and proportion of B.1.1.7. We assessed the Spearman correlation between the proportion of B.1.1.7 cases and number of reinfections over time, and between the number of positive tests and reinfections. We estimated incidence for B.1.1.7 and previous variants, and compared the effective reproduction number, Rt, for the two incidence estimates. Findings From Sept 28 to Dec 27, 2020, positive COVID-19 tests were reported by 36 920 COVID Symptom Study app users whose region was known and who reported as healthy on app sign-up. We found no changes in reported symptoms or disease duration associated with B.1.1.7. For the same period, possible reinfections were identified in 249 (0·7% [95% CI 0·6–0·8]) of 36 509 app users who reported a positive swab test before Oct 1, 2020, but there was no evidence that the frequency of reinfections was higher for the B.1.1.7 variant than for pre-existing variants. Reinfection occurrences were more positively correlated with the overall regional rise in cases (Spearman correlation 0·56–0·69 for South East, London, and East of England) than with the regional increase in the proportion of infections with the B.1.1.7 variant (Spearman correlation 0·38–0·56 in the same regions), suggesting B.1.1.7 does not substantially alter the risk of reinfection. We found a multiplicative increase in the Rt of B.1.1.7 by a factor of 1·35 (95% CI 1·02–1·69) relative to pre-existing variants. However, Rt fell below 1 during regional and national lockdowns, even in regions with high proportions of infections with the B.1.1.7 variant. Interpretation The lack of change in symptoms identified in this study indicates that existing testing and surveillance infrastructure do not need to change specifically for the B.1.1.7 variant. In addition, given that there was no apparent increase in the reinfection rate, vaccines are likely to remain effective against the B.1.1.7 variant. Funding Zoe Global, Department of Health (UK), Wellcome Trust, Engineering and Physical Sciences Research Council (UK), National Institute for Health Research (UK), Medical Research Council (UK), Alzheimer's Society

Genomic assessment of quarantine measures to prevent SARS-CoV-2 importation and transmission

Mitigation of SARS-CoV-2 transmission from international travel is a priority. We evaluated the effectiveness of travellers being required to quarantine for 14-days on return to England in Summer 2020. We identified 4,207 travel-related SARS-CoV-2 cases and their contacts, and identified 827 associated SARS-CoV-2 genomes. Overall, quarantine was associated with a lower rate of contacts, and the impact of quarantine was greatest in the 16–20 age-group. 186 SARS-CoV-2 genomes were sufficiently unique to identify travel-related clusters. Fewer genomically-linked cases were observed for index cases who returned from countries with quarantine requirement compared to countries with no quarantine requirement. This difference was explained by fewer importation events per identified genome for these cases, as opposed to fewer onward contacts per case. Overall, our study demonstrates that a 14-day quarantine period reduces, but does not completely eliminate, the onward transmission of imported cases, mainly by dissuading travel to countries with a quarantine requirement

Investigation of hospital discharge cases and SARS-CoV-2 introduction into Lothian care homes

Background The first epidemic wave of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Scotland resulted in high case numbers and mortality in care homes. In Lothian, over one-third of care homes reported an outbreak, while there was limited testing of hospital patients discharged to care homes. Aim To investigate patients discharged from hospitals as a source of SARS-CoV-2 introduction into care homes during the first epidemic wave. Methods A clinical review was performed for all patients discharges from hospitals to care homes from 1st March 2020 to 31st May 2020. Episodes were ruled out based on coronavirus disease 2019 (COVID-19) test history, clinical assessment at discharge, whole-genome sequencing (WGS) data and an infectious period of 14 days. Clinical samples were processed for WGS, and consensus genomes generated were used for analysis using Cluster Investigation and Virus Epidemiological Tool software. Patient timelines were obtained using electronic hospital records. Findings In total, 787 patients discharged from hospitals to care homes were identified. Of these, 776 (99%) were ruled out for subsequent introduction of SARS-CoV-2 into care homes. However, for 10 episodes, the results were inconclusive as there was low genomic diversity in consensus genomes or no sequencing data were available. Only one discharge episode had a genomic, time and location link to positive cases during hospital admission, leading to 10 positive cases in their care home. Conclusion The majority of patients discharged from hospitals were ruled out for introduction of SARS-CoV-2 into care homes, highlighting the importance of screening all new admissions when faced with a novel emerging virus and no available vaccine

SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway

Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron (B.1.1.529), the fifth VOC to be described, harbours multiple amino acid mutations in spike, half of which lie within the receptor-binding domain. Here we demonstrate substantial evasion of neutralization by Omicron BA.1 and BA.2 variants in vitro using sera from individuals vaccinated with ChAdOx1, BNT162b2 and mRNA-1273. These data were mirrored by a substantial reduction in real-world vaccine effectiveness that was partially restored by booster vaccination. The Omicron variants BA.1 and BA.2 did not induce cell syncytia in vitro and favoured a TMPRSS2-independent endosomal entry pathway, these phenotypes mapping to distinct regions of the spike protein. Impaired cell fusion was determined by the receptor-binding domain, while endosomal entry mapped to the S2 domain. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant