Deep learning-based virtual H& E staining from label-free autofluorescence lifetime images.

Label-free autofluorescence lifetime is a unique feature of the inherent fluorescence signals emitted by natural fluorophores in biological samples. Fluorescence lifetime imaging microscopy (FLIM) can capture these signals enabling comprehensive analyses of biological samples. Despite the fundamental importance and wide application of FLIM in biomedical and clinical sciences, existing methods for analysing FLIM images often struggle to provide rapid and precise interpretations without reliable references, such as histology images, which are usually unavailable alongside FLIM images. To address this issue, we propose a deep learning (DL)-based approach for generating virtual Hematoxylin and Eosin (H&E) staining. By combining an advanced DL model with a contemporary image quality metric, we can generate clinical-grade virtual H&E-stained images from label-free FLIM images acquired on unstained tissue samples. Our experiments also show that the inclusion of lifetime information, an extra dimension beyond intensity, results in more accurate reconstructions of virtual staining when compared to using intensity-only images. This advancement allows for the instant and accurate interpretation of FLIM images at the cellular level without the complexities associated with co-registering FLIM and histology images. Consequently, we are able to identify distinct lifetime signatures of seven different cell types commonly found in the tumour microenvironment, opening up new opportunities towards biomarker-free tissue histology using FLIM across multiple cancer types.

Cancer-associated fibroblasts expressing fibroblast activation protein and podoplanin in non-small cell lung cancer predict poor clinical outcome.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are a dominant cell type in the stroma of non-small cell lung cancer (NSCLC). Fibroblast heterogeneity reflects subpopulations of CAFs, which can influence prognosis and treatment efficacy. We describe the subtypes of CAFs in NSCLC. METHODS: Primary human NSCLC resections were assessed by flow cytometry and multiplex immunofluorescence for markers of fibroblast activation which allowed identification of CAF subsets. Survival data were analysed for our NSCLC cohort consisting of 163 patients to understand prognostic significance of CAF subsets. RESULTS: We identified five CAF populations, termed CAF S1-S5. CAF-S5 represents a previously undescribed population, and express FAP and PDPN but lack the myofibroblast marker αSMA, whereas CAF-S1 populations express all three. CAF-S5 are spatially further from tumour regions then CAF-S1 and scRNA data demonstrate an inflammatory phenotype. The presence of CAF-S1 or CAF-S5 is correlated to worse survival outcome in NSCLC, despite curative resection, highlighting the prognostic importance of CAF subtypes in NSCLC. TCGA data suggest the predominance of CAF-S5 has a poor prognosis across several cancer types. CONCLUSION: This study describes the fibroblast heterogeneity in NSCLC and the prognostic importance of the novel CAF-S5 subset where its presence correlates to worse survival outcome.

Fibre-based fluorescence-lifetime imaging microscopy: a real-time biopsy guidance tool for suspected lung cancer.

Lung cancer is the most common cause of cancer-related deaths worldwide. Early detection improves outcomes, however, existing sampling techniques are associated with suboptimal diagnostic yield and procedure-related complications. Autofluorescence-based fluorescence-lifetime imaging microscopy (FLIM), a technique which measures endogenous fluorophore decay rates, may aid identification of optimal biopsy sites in suspected lung cancer. Our fibre-based fluorescence-lifetime imaging system, utilising 488 nm excitation, which is deliverable via existing diagnostic platforms, enables real-time visualisation and lifetime analysis of distal alveolar lung structure. We evaluated the diagnostic accuracy of the fibre-based fluorescence-lifetime imaging system to detect changes in fluorescence lifetime in freshly resected ex vivo lung cancer and adjacent healthy tissue as a first step towards future translation. The study compares paired non-small cell lung cancer (NSCLC) and non-cancerous tissues with gold standard diagnostic pathology to assess the performance of the technique. Paired NSCLC and non-cancerous lung tissues were obtained from thoracic resection patients (N=21). A clinically compatible 488 nm fluorescence-lifetime endomicroscopy platform was used to acquire simultaneous fluorescence intensity and lifetime images. Fluorescence lifetimes were calculated using a computationally-lightweight, rapid lifetime determination method. Fluorescence lifetime was significantly reduced in ex vivo lung cancer, compared with non-cancerous lung tissue [mean ± standard deviation (SD), 1.79±0.40 vs. 2.15±0.26 ns, P<0.0001], and fluorescence intensity images demonstrated distortion of alveolar elastin autofluorescence structure. Fibre-based fluorescence-lifetime imaging demonstrated good performance characteristics for distinguishing lung cancer, from adjacent non-cancerous tissue, with 81.0% sensitivity and 71.4% specificity. Our novel fibre-based fluorescence-lifetime imaging system, which enables label-free imaging and quantitative lifetime analysis, discriminates ex vivo lung cancer from adjacent healthy tissue. This minimally invasive technique has potential to be translated as a real-time biopsy guidance tool, capable of optimising diagnostic accuracy in lung cancer.

Fibre-optic based exploration of lung cancer autofluorescence using spectral fluorescence lifetime.

Fibre-optic based time-resolved fluorescence spectroscopy (TRFS) is an advanced spectroscopy technique that generates sample-specific spectral-temporal signature, characterising variations in fluorescence in real-time. As such, it can be used to interrogate tissue autofluorescence. Recent advancements in TRFS technology, including the development of devices that simultaneously measure high-resolution spectral and temporal fluorescence, paired with novel analysis methods extracting information from these multidimensional measurements effectively, provide additional insight into the underlying autofluorescence features of a sample. This study demonstrates, using both simulated data and endogenous fluorophores measured bench-side, that the shape of the spectral fluorescence lifetime, or fluorescence lifetimes estimated over high-resolution spectral channels across a broad range, is influenced by the relative abundance of underlying fluorophores in mixed systems and their respective environment. This study, furthermore, explores the properties of the spectral fluorescence lifetime in paired lung tissue deemed either abnormal or normal by pathologists. We observe that, on average, the shape of the spectral fluorescence lifetime at multiple locations sampled on 14 abnormal lung tissue, compared to multiple locations sampled on the respective paired normal lung tissue, shows more variability; and, while not statistically significant, the average spectral fluorescence lifetime in abnormal tissue is consistently lower over every wavelength than the normal tissue.

Combined fluorescence lifetime and surface topographical imaging of biological tissue.

In this work a combined fluorescence lifetime and surface topographical imaging system is demonstrated. Based around a 126 × 192 time resolved single photon avalanche diode (SPAD) array operating in time correlated single-photon counting (TCSPC) mode, both the fluorescence lifetime and time of flight (ToF) can be calculated on a pixel by pixel basis. Initial tests on fluorescent samples show it is able to provide 4 mm resolution in distance and 0.4 ns resolution in lifetime. This combined modality has potential biomedical applications such as surgical guidance, endoscopy, and diagnostic imaging. The system is demonstrated on both ovine and human pulmonary tissue samples, where it offers excellent fluorescence lifetime contrast whilst also giving a measure of the distance to the sample surface.

Erratum to "Optical Detection of Distal Lung Enzyme Activity in Human Inflammatory Lung Disease".

[This corrects the article DOI: 10.34133/2021/9834163.].

Location of CD39+ T cell subpopulations within tumors predict differential outcomes in non-small cell lung cancer.

PURPOSE: An improved mechanistic understanding of immunosuppressive pathways in non-small cell lung cancer (NSCLC) is important to develop novel diagnostic and therapeutic approaches. Here, we investigate the prognostic significance of the ectonucleotidases CD39 and CD73 in NSCLC. EXPERIMENTAL DESIGN: The expression and localization of CD39, CD73 and CD103 was digitally quantified in a cohort of 162 early treatment naïve NSCLC patients using multiplex-immunofluorescence and related to patient outcome. Expression among different cell-populations was assessed via flow cytometry. Targeted RNA-Seq was performed on CD4+ and CD8+ T cells from digested NSCLC tumor tissue and single-cell RNA-Seq data was analyzed to investigate the functional significance of CD39+ T cell populations. RESULTS: We demonstrate that flow cytometry of early untreated NSCLC patients shows an upregulation of CD39 expression in the tumor tissue among natural killer (NK) cells, fibroblasts and T cells. CD73 expression is mainly found among fibroblasts and Epcam+cells in the tumor tissue. Multiplex Immunofluorescence in a cohort of 162 early untreated NSCLC patients demonstrates that CD39 expression is mainly localized in the tumor stroma while CD73 expression is equally distributed between tumor nest and stroma, and high expression of CD39 and CD73 in the tumor stroma is associated with poor recurrence-free survival (RFS) at 5 years. Additionally, we find that CD8+T cells located in the tumor nest express CD103 and the density of CD39+CD103+CD8+ T cells in the tumor nest predicts improved RFS at 5 years. Targeted RNA-Seq shows that the tumor microenvironment of NSCLC upregulates regulatory pathways in CD4+ T cells and exhaustion in CD8+ T cells, and analysis of a single cell RNA sequencing dataset shows that CD39+CD4+ cells are enriched in Treg signature gene-sets, and CD39+CD103+ cytotoxic T lymphocyte show gene signatures indicative of an exhausted cytotoxic phenotype with upregulated expression of CXCL13. CONCLUSIONS: Knowledge of patterns of distribution and location are required to understand the prognostic impact of CD39+ T cell populations in NSCLC. This study provides an improved understanding of spatial and functional characteristics of CD39+ T cells and their significance to patient outcome.

Quantitative proteomics identifies tumour matrisome signatures in patients with non-small cell lung cancer.

Introduction: The composition and remodelling of the extracellular matrix (ECM) are important factors in the development and progression of cancers, and the ECM is implicated in promoting tumour growth and restricting anti-tumour therapies through multiple mechanisms. The characterisation of differences in ECM composition between normal and diseased tissues may aid in identifying novel diagnostic markers, prognostic indicators and therapeutic targets for drug development. Methods: Using tissue from non-small cell lung cancer (NSCLC) patients undergoing curative intent surgery, we characterised quantitative tumour-specific ECM proteome signatures by mass spectrometry. Results: We identified 161 matrisome proteins differentially regulated between tumour tissue and nearby non-malignant lung tissue, and we defined a collagen hydroxylation functional protein network that is enriched in the lung tumour microenvironment. We validated two novel putative extracellular markers of NSCLC, the collagen cross-linking enzyme peroxidasin and a disintegrin and metalloproteinase with thrombospondin motifs 16 (ADAMTS16), for discrimination of malignant and non-malignant lung tissue. These proteins were up-regulated in lung tumour samples, and high PXDN and ADAMTS16 gene expression was associated with shorter survival of lung adenocarcinoma and squamous cell carcinoma patients, respectively. Discussion: These data chart extensive remodelling of the lung extracellular niche and reveal tumour matrisome signatures in human NSCLC.

Cancer-associated fibroblasts drive CXCL13 production in activated T cells via TGF-beta.

Introduction: Tumour-reactive T cells producing the B-cell attractant chemokine CXCL13, in solid tumours, promote development of tertiary lymphoid structures (TLS) and are associated with improved prognosis and responsiveness to checkpoint immunotherapy. Cancer associated fibroblasts are the dominant stromal cell type in non-small cell lung cancer (NSCLC) where they co-localise with T cells and can influence T cell activation and exhaustion. We questioned whether CAF directly promote CXCL13-production during T cell activation. Methods: We characterised surface markers, cytokine production and transcription factor expression in CXCL13-producing T cells in NSCLC tumours and paired non-cancerous lung samples using flow cytometry. We then assessed the influence of human NSCLC-derived primary CAF lines on T cells from healthy donors and NSCLC patients during activation in vitro measuring CXCL13 production and expression of cell-surface markers and transcription factors by flow cytometry. Results: CAFs significantly increased the production of CXCL13 by both CD4+ and CD8+ T cells. CAF-induced CXCL13-producing cells lacked expression of CXCR5 and BCL6 and displayed a T peripheral helper cell phenotype. Furthermore, we demonstrate CXCL13 production by T cells is induced by TGF-ß and limited by IL-2. CAF provide TGF-ß during T cell activation and reduce availability of IL-2 both directly (by reducing the capacity for IL-2 production) and indirectly, by expanding a population of activated Treg. Inhibition of TGF-ß signalling prevented both CAF-driven upregulation of CXCL13 and Treg expansion. Discussion: Promoting CXCL13 production represents a newly described immune-regulatory function of CAF with the potential to shape the immune infiltrate of the tumour microenvironment both by altering the effector-function of tumour infiltrating T-cells and their capacity to attract B cells and promote TLS formation.

Defining the road map to a UK national lung cancer screening programme.

Lung cancer screening with low-dose CT was recommended by the UK National Screening Committee (UKNSC) in September, 2022, on the basis of data from trials showing a reduction in lung cancer mortality. These trials provide sufficient evidence to show clinical efficacy, but further work is needed to prove deliverability in preparation for a national roll-out of the first major targeted screening programme. The UK has been world leading in addressing logistical issues with lung cancer screening through clinical trials, implementation pilots, and the National Health Service (NHS) England Targeted Lung Health Check Programme. In this Policy Review, we describe the consensus reached by a multiprofessional group of experts in lung cancer screening on the key requirements and priorities for effective implementation of a programme. We summarise the output from a round-table meeting of clinicians, behavioural scientists, stakeholder organisations, and representatives from NHS England, the UKNSC, and the four UK nations. This Policy Review will be an important tool in the ongoing expansion and evolution of an already successful programme, and provides a summary of UK expert opinion for consideration by those organising and delivering lung cancer screenings in other countries.

Computational Fluorescence Suppression in Shifted Excitation Raman Spectroscopy.

Fiber-based Raman spectroscopy in the context of in vivo biomedical application suffers from the presence of background fluorescence from the surrounding tissue that might mask the crucial but inherently weak Raman signatures. One method that has shown potential for suppressing the background to reveal the Raman spectra is shifted excitation Raman spectroscopy (SER). SER collects multiple emission spectra by shifting the excitation by small amounts and uses these spectra to computationally suppress the fluorescence background based on the principle that Raman spectrum shifts with excitation while fluorescence spectrum does not. We introduce a method that utilizes the spectral characteristics of the Raman and fluorescence spectra to estimate them more effectively, and compare this approach against existing methods on real world datasets.

Tri-mode optical biopsy probe with fluorescence endomicroscopy, Raman spectroscopy, and time-resolved fluorescence spectroscopy.

We present an endoscopic probe that combines three distinct optical fibre technologies including: A high-resolution imaging fibre for optical endomicroscopy, a multimode fibre for time-resolved fluorescence spectroscopy, and a hollow-core fibre with multimode signal collection cores for Raman spectroscopy. The three fibers are all enclosed within a 1.2 mm diameter clinical grade catheter with a 1.4 mm end cap. To demonstrate the probe's flexibility we provide data acquired with it in loops of radii down to 2 cm. We then use the probe in an anatomically accurate model of adult human airways, showing that it can be navigated to any part of the distal lung using a commercial bronchoscope. Finally, we present data acquired from fresh ex vivo human lung tissue. Our experiments show that this minimally invasive probe can deliver real-time optical biopsies from within the distal lung - simultaneously acquiring co-located high-resolution endomicroscopy and biochemical spectra.

An Inhaled Galectin-3 Inhibitor in COVID-19 Pneumonitis: A Phase Ib/IIa Randomized Controlled Clinical Trial (DEFINE).

Rationale: High circulating galectin-3 is associated with poor outcomes in patients with coronavirus disease (COVID-19). We hypothesized that GB0139, a potent inhaled thiodigalactoside galectin-3 inhibitor with antiinflammatory and antifibrotic actions, would be safely and effectively delivered in COVID-19 pneumonitis. Objectives: Primary outcomes were safety and tolerability of inhaled GB0139 as an add-on therapy for patients hospitalized with COVID-19 pneumonitis. Methods: We present the findings of two arms of a phase Ib/IIa randomized controlled platform trial in hospitalized patients with confirmed COVID-19 pneumonitis. Patients received standard of care (SoC) or SoC plus 10 mg inhaled GB0139 twice daily for 48 hours, then once daily for up to 14 days or discharge. Measurements and Main Results: Data are reported from 41 patients, 20 of which were assigned randomly to receive GB0139. Primary outcomes: the GB0139 group experienced no treatment-related serious adverse events. Incidences of adverse events were similar between treatment arms (40 with GB0139 + SoC vs. 35 with SoC). Secondary outcomes: plasma GB0139 was measurable in all patients after inhaled exposure and demonstrated target engagement with decreased circulating galectin (overall treatment effect post-hoc analysis of covariance [ANCOVA] over days 2-7; P = 0.0099 vs. SoC). Plasma biomarkers associated with inflammation, fibrosis, coagulopathy, and major organ function were evaluated. Conclusions: In COVID-19 pneumonitis, inhaled GB0139 was well-tolerated and achieved clinically relevant plasma concentrations with target engagement. The data support larger clinical trials to determine clinical efficacy. Clinical trial registered with ClinicalTrials.gov (NCT04473053) and EudraCT (2020-002230-32).

Understanding patient barriers and facilitators to uptake of lung screening using low dose computed tomography: a mixed methods scoping review of the current literature.

BACKGROUND: Targeted lung cancer screening is effective in reducing mortality by upwards of twenty percent. However, screening is not universally available and uptake is variable and socially patterned. Understanding screening behaviour is integral to designing a service that serves its population and promotes equitable uptake. We sought to review the literature to identify barriers and facilitators to screening to inform the development of a pilot lung screening study in Scotland. METHODS: We used Arksey and O'Malley's scoping review methodology and PRISMA-ScR framework to identify relevant literature to meet the study aims. Qualitative, quantitative and mixed methods primary studies published between January 2000 and May 2021 were identified and reviewed by two reviewers for inclusion, using a list of search terms developed by the study team and adapted for chosen databases. RESULTS: Twenty-one articles met the final inclusion criteria. Articles were published between 2003 and 2021 and came from high income countries. Following data extraction and synthesis, findings were organised into four categories: Awareness of lung screening, Enthusiasm for lung screening, Barriers to lung screening, and Facilitators or ways of promoting uptake of lung screening. Awareness of lung screening was low while enthusiasm was high. Barriers to screening included fear of a cancer diagnosis, low perceived risk of lung cancer as well as practical barriers of cost, travel and time off work. Being health conscious, provider endorsement and seeking reassurance were all identified as facilitators of screening participation. CONCLUSIONS: Understanding patient reported barriers and facilitators to lung screening can help inform the implementation of future lung screening pilots and national lung screening programmes.

Optimizing the implementation of lung cancer screening in Scotland: Focus group participant perspectives in the LUNGSCOT study.

INTRODUCTION: Targeted lung cancer screening is effective in reducing lung cancer and all-cause mortality according to major trials in the United Kingdom and Europe. However, the best ways of implementing screening in local communities requires an understanding of the population the programme will serve. We undertook a study to explore the views of those potentially eligible for, and to identify potential barriers and facilitators to taking part in, lung screening, to inform the development of a feasibility study. METHODS: Men and women aged 45-70, living in urban and rural Scotland, and either self-reported people who smoke or who recently quit, were invited to take part in the study via research agency Taylor McKenzie. Eleven men and 14 women took part in three virtual focus groups exploring their views on lung screening. Focus group transcripts were transcribed and analysed using thematic analysis, assisted by QSR NVivo. FINDINGS: Three overarching themes were identified: (1) Knowledge, awareness and acceptability of lung screening, (2) Barriers and facilitators to screening and (3) Promoting screening and implementation ideas. Participants were largely supportive of lung screening in principle and described the importance of the early detection of cancer. Emotional and psychological concerns as well as system-level and practical issues were discussed as posing barriers and facilitators to lung screening. CONCLUSIONS: Understanding the views of people potentially eligible for a lung health check can usefully inform the development of a further study to test the feasibility and acceptability of lung screening in Scotland. PATIENT OR PUBLIC CONTRIBUTION: The LUNGSCOT study has convened a patient advisory group to advise on all aspects of study development and implementation. Patient representatives commented on the focus group study design, study materials and ethics application, and two representatives read the focus group transcripts.

Deep learning-assisted co-registration of full-spectral autofluorescence lifetime microscopic images with H&E-stained histology images.

Autofluorescence lifetime images reveal unique characteristics of endogenous fluorescence in biological samples. Comprehensive understanding and clinical diagnosis rely on co-registration with the gold standard, histology images, which is extremely challenging due to the difference of both images. Here, we show an unsupervised image-to-image translation network that significantly improves the success of the co-registration using a conventional optimisation-based regression network, applicable to autofluorescence lifetime images at different emission wavelengths. A preliminary blind comparison by experienced researchers shows the superiority of our method on co-registration. The results also indicate that the approach is applicable to various image formats, like fluorescence in-tensity images. With the registration, stitching outcomes illustrate the distinct differences of the spectral lifetime across an unstained tissue, enabling macro-level rapid visual identification of lung cancer and cellular-level characterisation of cell variants and common types. The approach could be effortlessly extended to lifetime images beyond this range and other staining technologies.

Phototherapeutic Induction of Immunogenic Cell Death and CD8+ T Cell-Granzyme B Mediated Cytolysis in Human Lung Cancer Cells and Organoids.

Augmenting T cell mediated tumor killing via immunogenic cancer cell death (ICD) is the cornerstone of emerging immunotherapeutic approaches. We investigated the potential of methylene blue photodynamic therapy (MB-PDT) to induce ICD in human lung cancer. Non-Small Cell Lung Cancer (NSCLC) cell lines and primary human lung cancer organoids were evaluated in co-culture killing assays with MB-PDT and light emitting diodes (LEDs). ICD was characterised using immunoblotting, immunofluorescence, flow cytometry and confocal microscopy. Phototherapy with MB treatment and low energy LEDs decreased the proliferation of NSCLC cell lines inducing early necrosis associated with reduced expression of the anti-apoptotic protein, Bcl2 and increased expression of ICD markers, calreticulin (CRT), intercellular cell-adhesion molecule-1 (ICAM-1) and major histocompatibility complex I (MHC-I) in NSCLC cells. MB-PDT also potentiated CD8+ T cell-mediated cytolysis of lung cancer via granzyme B in lung cancer cells and primary human lung cancer organoids.

A fluorogenic probe for granzyme B enables in-biopsy evaluation and screening of response to anticancer immunotherapies.

Immunotherapy promotes the attack of cancer cells by the immune system; however, it is difficult to detect early responses before changes in tumor size occur. Here, we report the rational design of a fluorogenic peptide able to detect picomolar concentrations of active granzyme B as a biomarker of immune-mediated anticancer action. Through a series of chemical iterations and molecular dynamics simulations, we synthesize a library of FRET peptides and identify probe H5 with an optimal fit into granzyme B. We demonstrate that probe H5 enables the real-time detection of T cell-mediated anticancer activity in mouse tumors and in tumors from lung cancer patients. Furthermore, we show image-based phenotypic screens, which reveal that the AKT kinase inhibitor AZD5363 shows immune-mediated anticancer activity. The reactivity of probe H5 may enable the monitoring of early responses to anticancer treatments using tissue biopsies.

Cancer Associated Fibroblasts - An Impediment to Effective Anti-Cancer T Cell Immunity.

The presence of functionally efficient cytotoxic T lymphocytes (CTL) in the Tumour nest is crucial in mediating a successful immune response to cancer. The detection and elimination of cancer cells by CTL can be impaired by cancer-mediated immune evasion. In recent years, it has become increasingly clear that not only neoplastic cells themselves, but also cells of the tumour microenvironment (TME) exert immunosuppressive functions and thereby play an integral part in the immune escape of cancer. The most abundant stromal cells of the TME, cancer associated fibroblasts (CAFs), promote tumour progression via multiple pathways and play a role in dampening the immune response to cancer. Recent research indicates that T cells react to CAF signalling and establish bidirectional crosstalk that plays a significant role in the tumour immune response. This review discusses the various mechanisms by which the CAF/T cell crosstalk may impede anti-cancer immunity.

Fibroblast Activation Protein Specific Optical Imaging in Non-Small Cell Lung Cancer.

Fibroblast activation protein (FAP) is a cell surface propyl-specific serine protease involved in the regulation of extracellular matrix. Whilst expressed at low levels in healthy tissue, upregulation of FAP on fibroblasts can be found in several solid organ malignancies, including non-small cell lung cancer, and chronic inflammatory conditions such as pulmonary fibrosis and rheumatoid arthritis. Their full role remains unclear, but FAP expressing cancer associated fibroblasts (CAFs) have been found to relate to a poor prognosis with worse survival rates in breast, colorectal, pancreatic, and non-small cell lung cancer (NSCLC). Optical imaging using a FAP specific chemical probe, when combined with clinically compatible imaging systems, can provide a readout of FAP activity which could allow disease monitoring, prognostication and potentially stratify therapy. However, to derive a specific signal for FAP any sequence must retain specificity over closely related endopeptidases, such as prolyl endopeptidase (PREP), and be resistant to degradation in areas of active inflammation. We describe the iterative development of a FAP optical reporter sequence which retains FAP specificity, confers resistance to degradation in the presence of activated neutrophil proteases and demonstrates clinical tractability ex vivo in NSCLC samples with an imaging platform.