Boosting Cancer Immunotherapies with Optical Biosensor Nanotechnologies Authors: *Maria Soler,1 Laura M. Lechuga2 1. Networking Research Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain 2. Nanobiosensors and Bioanalytical Applications Group, Catalan Institute of Nanoscience and Nanotechnology (ICN2), UAB Campus, Bellaterra, Barcelona, Spain *Correspondence to maria.soler@icn2.cat Disclosure: The authors have declared no conflicts of interest. Acknowledgements: The authors acknowledge the financial support from PreDICT project (Programa estatal de investigación, desarrollo e innovación orientada a los Retos de la Sociedad, TEC2016-78515-R). ICN2 is the recipient of Grant SEV-2017-0706 from the “Severo Ochoa Centers of Excellence” Program of Spanish Ministry of Science. Received: 28.06.19 Accepted: 06.08.19 Keywords: Adoptive cell transfer, biomedical engineering, biosensors, cancer, cell analysis, immunotherapy, nanoplasmonics. Citation: EMJ. 2019;4[4]:124-132. Abstract An increasing number of cancer patients have found new hope in the form of recently discovered cell immunotherapies. These novel treatments empower our own immune system to fight and eradicate the malignant tumours, even in metastatic stages. Unprecedented outcomes have been demonstrated with bioengineered cells especially modified in laboratories to trigger a highly specific and active anti-tumour function; however, a main bottleneck for the implementation of these promising therapies resides in the old-fashioned and limited techniques employed for the in vitro analysis and study of cells during manufacturing and testing processes. Optical label-free biosensors are currently arising as promising analytical technologies for a simplified and accurate cell analysis. In particular, biosensors based on evanescent field principles enable direct, non-invasive, and real-time monitoring of biological events, showing outstanding sensitivities and excellent robustness. Furthermore, these biosensors are easily incorporated in lab-on-a-chip platforms for simple operation and implementation in clinical laboratories. In this review, the authors present and discuss the more recent research aimed to develop label-free optical biosensors for the analysis of live cells and the direct benefits offered to cell immunotherapy production allowing for its widespread implementation. THE REVOLUTION OF CANCER landmark in decades to come. While it has long been a pursued goal in immunology and IMMUNOTHERAPIES oncology, only now are the first clinical trials Cancer immunotherapy has been profiled as one showing significant and impressive results in of the major breakthroughs of the decade and the treatment of cancer patients.1 This relatively will probably remain a paramount biomedical new therapy seeks the activation, inhibition, 124 EUROPEAN MEDICAL JOURNAL • December 2019 EMJ EUROPEAN MEDICAL JOURNAL or improvement of the immune system to fight proteins or peptides solely or over-expressed in cancer. Instead of directly targeting tumour cancer cells. Several vaccination strategies are cells, such as conventional radiation therapy currently under development or being evaluated or chemotherapy, immunotherapy approaches in clinical and preclinical trials, including protein empower the patient’s natural immune system and peptide vaccines, genetic vaccines (DNA, to attack them. Therefore, the benefits promised RNA, and viral), and cell vaccines (tumour or by cancer immunotherapies include not only a immune cells). This therapeutic immunisation remarkable increase of treatment efficiencies, has demonstrated minimal toxicity in all clinical but also fewer side effects, and the possibility to studies and also offers a long-lasting protection treat certain cancer types that do not respond to against tumour appearance and progression. The other therapies or are in advanced stages. main challenge to cancer vaccines relies on the immunosuppressive tumour microenvironment Immunotherapy strategies can be categorised that prevents the immune cells to act and into three broad groups: passive proliferate and, therefore, suggests that vaccines immunomodulation, active immunisation (i.e., should be administered in combination with other cancer vaccines), and the adoptive cell transfer agents or approaches to synergistically engage approaches. The first passive immunomodulatory the anti-tumour response. treatments were approved by the U.S. Food and Drug Administration (FDA) in the 1990s and are Finally, one of the most attractive and widely available for cancer patients. This type of encouraging immunotherapies is the so-called immunotherapy consists of the administration adoptive cell therapy.4 This strategy consists of cytokines and other molecules that boost of identifying and/or engineering the patient’s the general immune response of the body to own T lymphocytes to enhance the anti-tumour attack the malignant cells. Among them, the activity, expand them ex vivo, and reinfuse them administration of IL-2, a T cell growth factor, is again into the patient with cancer (Figure 1). used to stimulate endogenous tumour-reactive The massive transfer of these powerful cells into cells in vivo and has shown reproducible the patient’s body has demonstrated an almost regression of solid human cancers. More recently, a immediate tumour regression and eradication new immunomodulatory approach has emerged, while offering a long-lasting protection against which makes use of monoclonal antibodies to cancer recurrence. Furthermore, because the inhibit the immune checkpoints that prevent T treatment employs the patient’s own cells, cells to identify and eliminate the tumour cells.2 the associated side effects and toxicity could The blockade of specific checkpoint molecules, be minimal. such as cytotoxic T-lymphocyte-associated 4 (CTLA4) or programmed-death 1 (PD1), have This article briefly reviews the current biomedical shown impressive results for the treatment of challenges associated with cell immunotherapies, different malignancies, such as bladder cancer, especially during the production and non-small cell lung cancer, and cutaneous evaluation processes, and how they can limit melanoma. In fact, the innovation of these their widespread administration to cancer approaches was awarded with the Nobel Prize patients. The authors also introduce optical in Medicine in 2018. However, because these biosensor nanotechnologies as pioneering and drugs interfere with the patient immune system powerful platforms to boost and facilitate the in a non-specific manner, the treatment is far manufacturing and implementation of such from reaching optimum efficacy and probably personalised medicine, enabling a highly causes severe side effects. accurate, faster, and simpler analysis of live cells and immune system activity. Instead, a more active immunotherapy could provide better outcomes. Therapeutic cancer vaccines are generally intended to treat growing CELL IMMUNOTHERAPIES: PROMISES tumours by strengthening the immune response AND CHALLENGES of the patient to selectively attack the malignant cells.3 For that purpose, the vaccines are There are two main types of cell-based designed to activate the T cells by presenting immunotherapies depending on whether they specific tumour-associated antigens, which are use the patient’s own tumour infiltrating Creative Commons Attribution-Non Commercial 4.0 December 2019 • EUROPEAN MEDICAL JOURNAL 125 lymphocytes (TIL) or artificially bioengineered T so far demonstrated outstanding response cells carrying a chimeric antigen receptor (CAR) rates in melanoma, even in metastatic stages. on the cell surface. Ongoing trials for breast, ovarian, or colorectal cancer are showing promising results when Autologous TIL are immune cells that have combined with costimulatory ligands or other recognised and infiltrated the tumour to immunotherapeutic strategies; however, the eradicate it but have been inactivated by broad applicability of TIL therapy is limited the effect of the tumour microenvironment because not all tumours yield to immunologically and cancer immunosuppressive strategies. active T cells and not all cancer patients have These lymphocytes can be extracted from TIL. Identifying antigen-specific T cells with the tumour peripheral blood, expanded effective anti-tumour function in blood samples ex vivo, and stimulated to reinforce their anti- is an arduous task in clinical and biomedical tumour immune activity.5 Prior to reinfusion, the laboratories. Another important limitation patient undergoes a previous lymphodepletion relates to the manufacturing process of the procedure to eliminate endogenous lymphocytes therapy. TIL are usually grown with multiple that can compete with activated TIL. Additionally, rounds of expansion using cytokines or other the cell transfer is usually aided by co-adjuvants, stimuli, and it has been shown that their immune such as IL-2, to enhance the anti-tumour activity and anti-tumour efficiency is seriously performance. The adoptive TIL therapy has reduced after the long-term ex vivo cultures. 1. Blood extraction and T 2. T cell activation by cell isolation genetic modification 5. Re-infusion into the patient 4. In vitro cloning and Analysis of anti-tumour expansion activity and specificity Figure 1. Schematic representation of the production process of cell immunotherapies based on adoptive cell transfer. 126 EUROPEAN MEDICAL JOURNAL • December 2019 EMJ EUROPEAN MEDICAL JOURNAL Reducing the number of expansion cycles during tumour activity before re-infusion into the patient therapy production is a key factor to enhance (Figure 1). Currently, the analysis of the live cell the final effectivity and performance of the functionality in clinical and research laboratories immune treatment. is based on fluorescent or colorimetric techniques, such as flow cytometry and An alternative strategy is providing the most enzyme-linked immunosorbent assays (ELISA). exciting results for cell immunotherapies. In the These methodologies have demonstrated so-called ‘CAR-T’ cell therapy, T lymphocytes from outstanding sensitivities for analysing and sorting the patients are modified with artificial surface cell phenotypes according to protein expression receptors (i.e., CAR) that consist of an antibody- and secretion; however, they experience derived ligand fused with the T cell receptor important constraints in terms of automation, signalling machinery.6 A CAR moiety therefore time resolution, cost efficiency, and cell viability. combines a specific antigen binding domain that The need for fluorescent and colorimetric confers recognition of a tumour-specific antigen, labels for the detection not only compromises with intracellular signalling motifs that promote cell activity and recovery after the study, but T cell activation. The main advantage of CAR-T also limits the analysis to endpoint results and cell therapy in comparison with the TIL strategy impedes the integration in appropriate culture is that the CAR-T cells can be bioengineered and environments. Techniques such as flow cytometry produced from a small number of T lymphocytes, require initial large amounts of cells, which usually independently of their native anti-tumour avidity. imply previous in vitro expansion procedures Two of these immunotherapies have been that reduce the eventual anti-tumour efficiency already approved by the FDA for treating blood of the cells and inevitably delay the therapy cancers (i.e., acute lymphoblastic leukaemia and administration to the patient. aggressive B-cell non-Hodgkin lymphoma). The European Medicines Agency (EMA) has also Herein, the introduction of optical biosensor started the approval procedure for these CAR-T nanotechnologies to the field of cell analysis cell therapies. In parallel, many laboratories could bring an opportunity for accelerating the worldwide are intensively working on developing, production process of cell immunotherapies, optimising, and evaluating new CAR-T cell improving the evaluation accuracies, and also therapies for attacking solid tumours; however, reducing the final costs of the treatment. relevant success has not yet been accomplished, probably due to the multiple immunosuppressive OPTICAL BIOSENSORS FOR LABEL- mechanisms that T cells find within the tumour FREE AND REAL-TIME ANALYSIS microenvironment. As well as this, CAR-T cell therapy has been associated with cytokine Optical biosensors exploit the electromagnetic release syndrome in patients. The rapid activation properties of light for the detection, and expansion of these T cells upon infusion quantification, and analysis of biochemical may trigger an immune system reaction that interactions with a high sensitivity, and with can yield to fever, organ dysfunction, neurologic capabilities for label-free and real-time format symptoms, and other toxicities. To manage these assays.9,10 Optical label-free biosensors are serious side effects, the use of corticosteroids generally based on the evanescent wave or cytokine receptor blockers along with the techniques, which enables the detection of minute immunotherapy is being tested, with promising refractive index variations caused by changes of results so far. mass within an evanescent electromagnetic field Beyond the particular biomedical challenges, that is generated at the interface between the cell immunotherapies need to face sensor and the sample (100–500 nm). The two important obstacles that hamper their most common configurations are the surface widespread implementation and administration.7,8 plasmon resonance (SPR)-based biosensors The manufacturing and evaluation process of and the waveguide-based technologies, such cell immunotherapies involves multiple and as resonators or interferometers (Figure 2). SPR highly specialised procedures, including labour- systems employ a nanometre thin layer of gold intensive cultures and routine performance of cell (45–50 nm) as sensor surface. Upon illumination analyses to verify immune responses and anti- at certain angle and wavelength, the free Creative Commons Attribution-Non Commercial 4.0 December 2019 • EUROPEAN MEDICAL JOURNAL 127 A) Surface plasmon resonance biosensor B) Nanoplasmonic biosensors Evanescent field Localised evanescent field Analytes Receptors Gold Prism Light source Detector Light source Detector C) Resonant waveguide grating biosensor D) Waveguide interometer biosensors Evanescent field Evanescent field Analytes Analytes Receptors Receptors Waveguide Waveguide Grating Reference Light source Detector Figure 2: (A) Illustration of a surface plasmon resonance biosensor working in prism-coupling configuration. (B) Illustration of a localised surface plasmon resonance biosensor working in prism-coupling configuration. Right panels show scanning electron microscopy images of different nanoplasmonic structures. (C) Illustration of a resonant waveguide grating biosensor in angular configuration. (D) Illustration of a waveguide interferometer biosensor. electrons of the metal start oscillating collectively arose. Nanoplasmonic biosensors incorporate creating the so-called plasmon resonance, nanostructured surfaces such as nanodiscs, which in turn generates an evanescent wave nanorods, nanoslits, and nanoholes that exhibit a that penetrates in the dielectric medium and localised surface plasmon resonance and provide propagates a small distance along the surface. exceptional features that can be translated in enhanced sensitivities, multiplexing capabilities, The plasmon resonance is characterised by the or higher miniaturisation and integration appearance of an absorption dip in the reflected light. By measuring variations in the angle, adaptability.10,15,16 On the other hand, waveguide- wavelength, or intensity of the SPR dip, it is possible based technologies are based on the leaky to monitor in real time biological events occurring electromagnetic mode of light travelling along on the sensor surface.11 SPR biosensors are a waveguide material. The resonant waveguide widely commercially available and are employed grating (RWG) biosensors are commonly used in many research laboratories and pharmaceutical and commercially available. In this platform, industries to carry out routine analysis of the light is coupled to a waveguide via grating biomolecular interactions, kinetic analysis, or drug structures, where it propagates through discovery and evaluation assays.11–14 Accompanied generating an evanescent field and resulting by the progress in nanotechnology in the last in a narrowband of reflected or transmitted decade, more sophisticated versions of plasmonic wavelengths detected at the output.17 RWG biosensors have appeared and exponentially biosensors have been successfully integrated in 128 EUROPEAN MEDICAL JOURNAL • December 2019 EMJ EUROPEAN MEDICAL JOURNAL multiwell plates and imaging configuration for and tumour response. The high sensitivity, label- high-throughput and multiplexed screening.18 free format, and real-time analysis capabilities of In parallel to RWG systems, waveguide-based optical biosensors might enable the rapid isolation interferometers have also been realised, of specific T lymphocytes with anti-tumour achieving outstanding sensitivities for label- reactivity directly from tumour peripheral blood free detection. Here, the measuring guided samples, without the need for previous expansion light mode, in contact with the sample, is steps and avoiding cell staining with fluorescent combined to a reference light creating an markers. On the other hand, retrieving real-time interference. The differences in phase are used and quantitative information of cell activity to detect and quantify biochemical interactions events, such as membrane receptor activity or occurring at the sensor area.19 Some examples protein secretion, provides direct insights in cell of waveguide-based sensors are the well-known status and functionality. Mach-Zehnder interferometers,20 the bimodal waveguide interferometers,21 or the grating- For example, cancer cells secrete cytokines to coupled interferometers.22 the tumour microenvironment in order to grow and proliferate, or to inhibit the action of immune Over the last years, there has been a steady cells trying to attack them. Likewise, immune increase in the number of scientific publications T cells can release different types of proteins and patents in the area of optical label-free (e.g., cytokines, interferons, necrosis factors, and biosensors for the detection of numerous enzymes) for either killing tumour cells or as a molecules or pathogens, such as bacteria, signalling tool for recruiting other immune cells viruses, DNA or RNA, proteins, peptides, or small and enhancing the immune response. Thus, molecules like drugs or neurotransmitters.10,11,19,23 the label-free and real-time analysis of cell Surprisingly, the application of optical label-free secretion can facilitate the evaluation of immune platforms for live cell biology remains relatively cell activation upon interaction with tumour unexplored.24,25 The predominate challenges antigens, the cancer cell response, or the study are related to the lack of an appropriate of immunosuppressive strategies in tumour biochemical design for providing clinically relevant microenvironments (Figure 3A). For the study of information; the use of conventional microfluidic cancer cell activity and tumour progression, Liu systems being unsuitable for cell culture and et al.26 developed a SPR biosensor able to monitor, long-term analysis; and the inherent limitations in real time, the secretion of vascular endothelial of mass-related sensing of optical label-free growth factor (VEGF), a cytokine responsible for methods, which commonly yields to insufficient promoting the formation of blood vessels and sensitivity for small biomolecule detection and tumour spread. The SPR system incorporated a selectivity issues when evaluating complex mini cell culture module within the microfluidics fluids, such as culture media or blood serum that was placed close to the sensor surface. and plasma. Nevertheless, a few studies have Ovarian carcinoma cells were stimulated with investigated the use of label-free optical sensing a calcium ionophore and the secretion of VEGF technology for the analysis of cell functionality was readily detected by specific antibodies in real time, showing promising potential, immobilised on the sensor surface. Li et al.27 especially for the implementation in cell developed a more advanced lab-on-a-chip immunotherapy development. nanoplasmonic system for VEGF secretion analysis from live HeLa cells. The platform OPTICAL LABEL-FREE consisted of two interconnected modules: a BIOSENSORS FOR CELL microfluidic cell module, where cells could be IMMUNOTHERAPY APPLICATIONS cultured with appropriate temperature and humidity conditions, and the optical sensor Two critical steps in the cell immunotherapy module based on gold nanohole arrays. The development process could be greatly improved nanoplasmonic sensor with multiplexing with the introduction of optical sensing adaptability monitored the culture cell technologies: the detection and identification supernatant for up to 10 hours and was able of relevant immune cells from human samples, to detect VEGF secretion from cancer cells at and the evaluation of the immune cell activity approximately 6 hours after stimulation. Creative Commons Attribution-Non Commercial 4.0 December 2019 • EUROPEAN MEDICAL JOURNAL 129 (A) Cell secretion assay (B) Dynamic mass (C) Cell detection and redistribution assay capturing assay Cytokines Signalling cascade Tumour Membrane Antibodies Stimuli or Membrane receptors receptors antigens inhibitors Figure 3: Conceptual illustrations of different cell assays performed with optical label-free biosensors. (A) Detection of protein secretion; (B) Detection of cytosolic dynamic mass redistribution (DMR); (C) Detection and capture of specific anti-tumour cells. For the analysis of immune cell activity, some gated microfluidic chamber to monitor IL-2 promising approaches have also been proposed. secretion from isolated T cells. The optofluidic Oh et al.28 demonstrated for the first time the system enabled not only the ultrasensitive detection of cytokine secretion from immune T detection of the cytokine secretion, but also the cells directly isolated from human blood. Their spatio-temporal profiling of the secretion event, system was based on a nanoplasmonic surface revealing large heterogeneity and behaviour surrounded by a microfluidic cell chamber differences between various cells from the same only separated by a micropillar array. After cell cell line. incubation and stimulation, they could monitor the detection of TNF-α directly secreted by CD45 Another approach for elucidating cell activity is cells. With this platform, they could perform a by measuring the triggering of membrane cellular function immunoanalysis with minimal receptor signalling. When live cells are placed blood sample volume (3 µL) and a total assay on the sensor surface, the evanescent field is time three times shorter than conventional able to probe changes of mass distribution ELISA. Later, the same group demonstrated inside the cytosol of the cell (i.e., dynamic the multiplexed analysis of different cytokines mass redistribution), such as those occurring (IL-2, IFN-γ, and TNF-α) employing a gold when the signalling is activated (Figure 3B). nanorod-based biosensor.29 Although in this This strategy, however, requires the evanescent system the cells were not cultured in situ, field to propagate over large areas ensuring they proved the potential of nanoplasmonic maximum coverage of the cell entity; therefore, biosensors for immune cell profiling in a rapid waveguide-based biosensors are better suited and accurate mode. Pursuing the sensitivity for this application. Fang et al.32,33 employed a limits of nanoplasmonics, researchers have also RWG biosensor with angular modulation to attempted single-cell secretion analysis. Raphael monitor the EGF-receptor activation in cancer et al.30 developed a gold nanodisc biosensor cells. Upon stimulation with EGF molecules, they and showed real-time detection of antibody were able to detect mass redistribution inside secretion directly from an individual hybridoma living cells in real time, which was attributed to cell. More recently, Li et al.31 employed a gold the molecular rearrangements in the cell during nanohole array sensor integrated with a valve- the signalling cascade; however, because EFG 130 EUROPEAN MEDICAL JOURNAL • December 2019 EMJ EUROPEAN MEDICAL JOURNAL stimulation is known to lead to cell rounding CONCLUSIONS AND FUTURE and detachment, it is also conceivable that the PERSPECTIVES biosensor was monitoring cell detachment and re-adhesion to the surface. The same approach Cell immunotherapies based on adoptive cell was employed by Kurucz et al.34 to monitor the transfer approaches have developed huge response of human B cells upon stimulation and expectations as novel solutions for efficient inhibition of immune receptors. In this case, non- cancer treatment and eradication. There adherent B cells were immobilised on the surface are however still considerable challenges via electrostatic interactions and the dynamic to be addressed in order to guarantee their mass redistribution was measured when cells widespread administration safely, timely, and were stimulated with B-cell receptor antibodies, at an acceptable cost. Among others, the as well as stimulation inhibitors. manufacturing process of cell immunotherapies Finally, compared to cell activity analysis, needs to be updated, incorporating modern cell analysis techniques that allow for a higher capturing and isolating specific immune cells automation in the production process and from complex samples is far more challenging in enhancement of the accuracy and reliability of optical label-free biosensors. In order to selectively cell biology studies. Optical label-free biosensors capture tumour-reactive immune cells, it is are now being profiled as a unique alternative necessary to employ specific tumour receptors to traditional cell analysis techniques. These as biorecognition elements. Unfortunately, the biosensor nanotechnologies have demonstrated binding strength between T cells and tumour capabilities for the real-time identification receptors is rather weak, compared to, for and monitoring of live cell activity without instance, antibody-antigen interactions (Figure amplification or staining procedures, enabling 3C). In this regard, Soler et al.35 developed a the evaluation of cell immune responses in a few biomimetic SPR biosensor in a recent study hours and only using a few cells. Such features for the detection of T cells through interaction could therefore eliminate several cell culture and with tumour-specific receptors (Figure 3). By expansion steps in the manufacturing process, monitoring the interaction in real time, they were reducing the costs and time delay from months able to extract kinetic and affinity parameters to days. Further investigation might focus on and identify tumour-reactive T cells that could be developing new lab-on-a-chip platforms that further employed for immunotherapy production. combine the recent advances in nanophotonics, microfluidics, and bioengineering to eventually deliver more robust, simple, and reliable devices for cell analysis. The upcoming synergy between nanotechnology and biomedical research may be the key for groundbreaking progress and promotion of cell immunotherapies, holding a bright potential for effective and personalised cancer medicine. References 1. Wang RF. A special issue on cancer 4. Rosenberg SA et al. Adoptive cell Nephrology. 2017;1(3):151-5. immunotherapy. Cell Res. 2017;27(1):1- transfer: A clinical path to effective cancer immunotherapy. Nat Rev 7. 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