Cancer Immunotherapy Studies Research Articles

New Metabolomic Insights Into Cancer.

Cancer has been marked by metabolic irregularities that fuel various aggressive activities such as rapid cell proliferation, evasion of the immune system, and spread to distant organs. Therefore, exploiting cancer metabolism for diagnosis, monitoring, or treatment has been extensively studied in the past couple of decades with various molecular and cellular techniques. More recently, investigating cancer diagnostics and treatments through advanced metabolomics has emerged, and these comprehensive approaches provide a holistic understanding of cancer metabolism, which supported the discovery of metabolic targets relevant across multiple cancer types and the development of more effective treatments. This study offers highlights of new knowledge on cancer metabolism enabled by recent metabolomics studies and their potential applications in aiding cancer research and predicting cancer treatment outcomes. Specifically, we discussed the use of advanced metabolomics in cancer metabolism, tumor microenvironment, and cancer immunotherapy studies to provide valuable insights that can shape future research efforts in the dynamic field of cancer metabolism research.

Direct comparison of canine and human immune responses using transcriptomic and functional analyses

The canine spontaneous cancer model is increasingly utilized to evaluate new combined cancer immunotherapy approaches. While the major leukocyte subsets and phenotypes are closely related in dogs and humans, the functionality of T cells and antigen presenting cells in the two species has not been previously compared in detail. Such information would be important in interpreting immune response data and evaluating the potential toxicities of new cancer immunotherapies in dogs. To address this question, we used in vitro assays to compare the transcriptomic, cytokine, and proliferative responses of activated canine and human T cells, and also compared responses in activated macrophages. Transcriptomic analysis following T cell activation revealed shared expression of 515 significantly upregulated genes and 360 significantly downregulated immune genes. Pathway analysis identified 33 immune pathways shared between canine and human activated T cells, along with 34 immune pathways that were unique to each species. Activated human T cells exhibited a marked Th1 bias, whereas canine T cells were transcriptionally less active overall. Despite similar proliferative responses to activation, canine T cells produced significantly less IFN-γ than human T cells. Moreover, canine macrophages were significantly more responsive to activation by IFN-γ than human macrophages, as reflected by co-stimulatory molecule expression and TNF-α production. Thus, these studies revealed overall broad similarity in responses to immune activation between dogs and humans, but also uncovered important key quantitative and qualitative differences, particularly with respect to T cell responses, that should be considered in designing and evaluating cancer immunotherapy studies in dogs.

Machine learning-based natural language processing to extract PD-L1 expression levels from clinical notes.

Introduction: PD-L1 expression is used to determine oncology patients' response to and eligibility for immunologic treatments; however, PD-L1 expression status often only exists in unstructured clinical notes, limiting ability to use it in population-level studies. Methods: We developed and evaluated a machine learning based natural language processing (NLP) tool to extract PD-L1 expression values from the nationwide Veterans Affairs electronic health record system. Results: The model demonstrated strong evaluation performance across multiple levels of label granularity. Mean precision of the overall PD-L1 positive label was 0.859 (sd, 0.039), recall 0.994 (sd, 0.013), and F1 0.921 (0.024). When a numeric PD-L1 value was identified, the mean absolute error of the value was 0.537 on a scale of 0 to 100. Conclusion: We presented an accurate NLP method for deriving PD-L1 status from clinical notes. By reducing the time and manual effort needed to review medical records, our work will enable future population-level studies in cancer immunotherapy.

In vitro models to study natural killer cell dynamics in the tumor microenvironment.

Immunotherapy is revolutionizing cancer therapy. The rapid development of new immunotherapeutic strategies to treat solid tumors is posing new challenges for preclinical research, demanding novel in vitro methods to test treatments. Such methods should meet specific requirements, such as enabling the evaluation of immune cell responses like cytotoxicity or cytokine release, and infiltration into the tumor microenvironment using cancer models representative of the original disease. They should allow high-throughput and high-content analysis, to evaluate the efficacy of treatments and understand immune-evasion processes to facilitate development of new therapeutic targets. Ideally, they should be suitable for personalized immunotherapy testing, providing information for patient stratification. Consequently, the application of in vitro 3-dimensional (3D) cell culture models, such as tumor spheroids and organoids, is rapidly expanding in the immunotherapeutic field, coupled with the development of novel imaging-based techniques and -omic analysis. In this paper, we review the recent advances in the development of in vitro 3D platforms applied to natural killer (NK) cell-based cancer immunotherapy studies, highlighting the benefits and limitations of the current methods, and discuss new concepts and future directions of the field.

Assessing surrogacy using restricted mean survival time ratio for overall survival in liver cancer immunotherapy studies.

e16222 Background: Recently, immunotherapy has played a crucial role in treating liver cancer, one of the major cancers that contributes to global cancer burden. Overall survival (OS) is widely applied in cancer trials to evaluate the treatment effects of new therapies. However, it requires more patients and longer follow-up time comparing with progression-free survival (PFS). In addition, while assessing the treatment effects of cancer immunotherapy, proportional hazard (PH) assumption is often violated due to issues such as delayed treatment effects. Restricted Mean Survival Time (RMST) ratio is increasingly used for treatment effect evaluation when the PH assumption is violated. Such change prompts an important question whether the surrogacy value of PFS will be affected when RMST ratio is used to characterize treatment effect. The aim of this study is to examine the feasibility of using PFS as a surrogate endpoint for OS when the treatment effect is measured using hazard ratio (HR) versus RMST ratio. Methods: The surrogacy of PFS on OS was evaluated through examining the association between PFS and OS using HR and RMST ratio. Seven immunotherapy studies published between 2000 and 2021 were included (Table). Information of examined studies such as treatment arms information, OS and PFS were collected. RMST ratio for PFS and OS were calculated based on the Kaplan-Meier plots extracted from each article using WebPlotDigitizer 4.4. The weighted least square regression lines and R^2 between OS and PFS for HR and RMST ratio were calculated. Results: Among 7 immunotherapy studies, 4 gave placebos to the control arms as treatments. 2 provided Sorafenib and 1 assigned the same drug as treatment arm but with different schedule. All 7 studies had OS and PFS as the endpoints. 2 studies violated the PH assumptions. Based on the data extracted from examined articles, a moderate correlation (0.52) between PFS and OS was observed for HR while low correlation (0.10) was observed for RMST ratio. Conclusions: The R^2 values differ greatly depending on whether HR or RMST ratio was used for assessing surrogacy. Our finding may have important implications for the design of future immunotherapy liver cancer trials. For future work, increasing the number of included studies for a more comprehensive analysis is needed. Moreover, trial-level surrogacy analysis should be conducted to complement the study-level investigation.[Table: see text]

Programmed death-ligand 1 expression on CD22-specific chimeric antigen receptor-modified T cells weakens antitumor potential.

The molecules of programmed cell death protein‐1 (PD‐1) and ligand‐1 (PD‐L1) become new therapeutic targets for cancer therapy. Although tumor‐expressed PD‐L1 molecule is frequently dispensable for checkpoint blockade in some cancer patients, recent studies suggest that T cell‐expressed PD‐L1 molecule might play a crucial role in antitumor immunity. Here, to investigate CD22 chimeric antigen receptor (CAR)‐T cell therapy, we have generated the different CD22 CAR‐T constructs. We noticed that tumor cells induced PD‐L1 expression on the surface of CD22 CAR‐T cells. The induced PD‐L1 might limit immunogenic responses of CAR‐T cells. T cell‐expressed PD‐L1 leads to a suppressive signal by PD‐1/PD‐L1 engagement of CD22 CAR‐T cells. Meanwhile, PD‐L1 suppresses CD22 CAR‐T cell differentiation into memory T cells and negatively affected secretions of several essential cytokines, such as interleukin‐2 (IL‐2) and tumor necrosis factor (TNF)‐α. We further observed that anti‐PD‐L1 monoclonal antibodies rescued cytokine secretion of CD22 CAR‐T cells rather than anti‐PD‐1 monoclonal antibodies. Our current studies provide a potential mechanism to understand the functions and roles of T cell‐expressed PD‐L1 in tumor microenvironment. These results will encourage the physicians to re‐recognize the important roles of PD‐L1 in cancer immunotherapy studies and provide the helpful guidance for clinical operation of PD‐L1 inhibition drugs.

Assessing surrogacy using restricted mean survival time ratio for overall survival in non-small cell lung cancer immunotherapy studies.

Proportional hazards (PH) assumption is often violated in cancer immunotherapy studies. Restricted mean survival time (RMST) ratio is a valid metric to quantify the size of treatment effect when non-proportional hazard (NPH) is present. This study investigated the use of RMST ratio and hazard ratio (HR) in studying progression-free survival (PFS) as a surrogate endpoint for overall survival (OS) in non-small cell lung cancer immunotherapy trials. Trial level data were collected from 14 phase III trials published between 2012 and 2018. A weighted least-square regression (WLSR) was performed to evaluate the trial-level surrogacy. Surrogacy was evaluated via the association between RMST ratios for PFS and OS and between HRs for PFS and OS. Using data extracted from published articles, low to moderate correlation (0.49) between PFS and OS was observed for HR while low correlation (0.35) was observed for RMST ratio. When trials violating PH in PFS were included, more consistent correlations for both HR (0.43) and RMST ratio (0.44) were observed. In summary, the strength of PFS surrogacy for OS depends on whether HR or RMST ratio are chosen. RMST ratio and additional sensitivity analysis should be considered in addition to HR.

Tumor Antigen-Based Nanovaccines for Cancer Immunotherapy: A Review.

As an important means of tumor immunotherapy, tumor vaccines have achieved exciting results in the past few decades. However, there are still many obstacles that hinder tumor vaccines from achieving maximum efficacy, including lack of tumor antigens, low antigen immunogenicity and poor delivery efficiency. To overcome these challenges, researchers have developed and investigated various new types of tumor antigens with higher antigenic specificity and broader antigen spectrum, such as tumor-specific peptide antigens, tumor lysates, tumor cell membrane, tumor associated exosomes, etc. At the same time, different nanoparticulate delivery platforms have been developed to increase the immunogenicity of the tumor antigens, for example by increasing their targeting efficiency of antigen-presenting cells and lymph nodes, and by co-delivering antigens with adjuvants. In this review, we summarized different types of the tumor antigens that have been reported, and introduced several nanovaccine strategies for increasing the immunogenicity of tumor antigens. The review of recent progress in these fields may provide reference for the follow-up studies of tumor antigen-based cancer immunotherapy.

Rapid, label-free classification of tumor-reactive T cell killing with quantitative phase microscopy and machine learning

Quantitative phase microscopy (QPM) enables studies of living biological systems without exogenous labels. To increase the utility of QPM, machine-learning methods have been adapted to extract additional information from the quantitative phase data. Previous QPM approaches focused on fluid flow systems or time-lapse images that provide high throughput data for cells at single time points, or of time-lapse images that require delayed post-experiment analyses, respectively. To date, QPM studies have not imaged specific cells over time with rapid, concurrent analyses during image acquisition. In order to study biological phenomena or cellular interactions over time, efficient time-dependent methods that automatically and rapidly identify events of interest are desirable. Here, we present an approach that combines QPM and machine learning to identify tumor-reactive T cell killing of adherent cancer cells rapidly, which could be used for identifying and isolating novel T cells and/or their T cell receptors for studies in cancer immunotherapy. We demonstrate the utility of this method by machine learning model training and validation studies using one melanoma-cognate T cell receptor model system, followed by high classification accuracy in identifying T cell killing in an additional, independent melanoma-cognate T cell receptor model system. This general approach could be useful for studying additional biological systems under label-free conditions over extended periods of examination.

Towards the rational design of a next-generation dendritic cell vaccine for cancer immunotherapy

As professional antigen presenting cells (APCs) capable of eliciting primary immune responses among naïve T cells, dendritic cells (DCs) offer an attractive target for immune intervention. While some strategies for vaccination have sought to deliver antigens direct to DCs&nbsp;<em>in vivo</em>, others have pulsed DCs with target antigens&nbsp;<em>ex vivo</em>&nbsp;prior to administration. Indeed, numerous clinical studies of cancer immunotherapy have been conducted over the past two decades based on this approach, most of them benefitting from the ease with which DCs may be differentiated&nbsp;<em>in vitro</em>&nbsp;from the peripheral blood monocytes of individual patients. Nevertheless, while therapies exploiting monocyte-derived DCs (moDCs) have been shown to be safe, clinical outcomes have been disappointing, efficacy having been limited by factors including the type of DCs used and the source of tumor antigens. Here we review recent developments in identifying DC subsets with more favorable properties for use in cancer vaccination, with particular emphasis on CD141⁺&nbsp;DCs capable of antigen cross-presentation and discuss alternative sources, such as induced pluripotent stem cells (iPSCs), amenable to manufacture at scale. Furthermore, we assess how different sources of tumor antigens may complement this approach for the design of next generation DC vaccines.

Development of humanized mouse with patient‐derived xenografts for cancer immunotherapy studies: A comprehensive review

Immunotherapy has revolutionized cancer treatment, however, not all tumor types and patients are completely responsive to this approach. Establishing predictive pre‐clinical models would allow for more accurate and practical immunotherapeutic drug development. Mouse models are extensively used as in vivo system for biomedical research. However, due to the significant differences between rodents and human, it is impossible to translate most of the findings from mouse models to human. Pharmacological development and advancing personalized medicine using patient‐derived xenografts relies on producing mouse models in which murine cells and genes are substituted with their human equivalent. Humanized mice (HM) provide a suitable platform to evaluate xenograft growth in the context of a human immune system. In this review, we discussed recent advances in the generation and application of HM models. We also reviewed new insights into the basic mechanisms, pre‐clinical evaluation of onco‐immunotherapies, current limitations in the application of these models as well as available improvement strategies. Finally, we pointed out some issues for future studies.

Oxygen regulation of TET enzymes.

Hypoxia has a significant impact on many physiological and pathological processes. Over the recent years, its role in modulation of epigenetic remodelling has also become clearer. In cancer, low oxygen environments and aberrant epigenomes often go hand in hand, and changes in DNA methylation are now commonly recognised as potential outcome indicators. TET (ten-eleven translocation) family enzymes are alpha-ketoglutarate-, iron- and oxygen-dependent DNA demethylases and are key players in these processes. Although TETs have historically been considered tumour suppressors, recent studies suggest that their functions in cancer might not be straightforward. Recently, inhibition of TETs has been reported to have positive impact in cancer immunotherapy and vaccination studies. This underlines the current interest in developing targeted pharmaceutical inhibitors of these enzymes. Here, we will survey the complexity of TET roles in cancer, and its hypoxic modulation, as well as highlight the potential of these enzymes as therapeutic targets.

The progress and challenge of anti-PD-1/PD-L1 immunotherapy in treating non-small cell lung cancer.

The use of programmed cell-death protein 1 (PD-1)/programmed cell-death ligand 1 (PD-L1) inhibitors is the standard therapy for the first-line or second-line treatment of patients with non-small-cell lung cancer (NSCLC). In contrast to current traditional treatments such as chemotherapy or radiotherapy, anti-PD-1 and anti-PD-L1 treatments can directly attenuate tumour-mediated exhaustion and effectively modulate the host anti-tumour immune response in vivo. In addition, compared with traditional therapy, PD-1/PD-L1 inhibitor monotherapy can significantly prolong survival without obvious side effects in the treatment of advanced NSCLC. Ideally, several biomarkers could be used to monitor the safety and effectiveness of anti-PD-1 and anti-PD-L1 treatments; however, the current lack of optimal prognostic markers remains a widespread limitation and challenge for further clinical applications, as does the possibility of immune-related adverse events and drug resistance. In this review, we aimed to summarise the latest progress in anti-PD-1/anti-PD-L1 treatment of advanced NSCLC, worldwide, including in China. An exploration of underlying biomarker identification and future challenges will be discussed in this article to facilitate translational studies in cancer immunotherapy.

Abstract 1339: Transcriptomic profiles improve characterization natural killer cells and predict response to cytokine therapy in clinical trial for dogs with pulmonary metastases

Abstract Introduction: Natural killer (NK) cells are key effectors of the innate immune system, but major differences between human and murine NK cells have been a barrier to translation. Outbred dogs are an important link for NK-based cancer immunotherapy studies, so better characterization of canine NK cells is needed. We used RNAseq to compare gene expression profiles of ex vivo dog NK cells to in vivo NK signatures from dogs with pulmonary metastases receiving inhaled recombinant human (rh) IL-15 in a phase I clinical trial. Methods: Eight dogs with spontaneous pulmonary metastases were enrolled on an IACUC and clinical trials review board-approved Phase I clinical trial of inhaled rhIL-15 using a 3+3 cohort design with escalating doses of inhaled rhIL-15. Blood was collected from study subjects immediately pre-treatment and on days 7, 14 and 21 after treatment initiation for isolation of NK cells (CD5dim) and RNAseq. We performed differential gene expression (DGE) comparing trial patients to healthy beagle CD5dim NK populations (resting) and ex vivo activated beagle NK cells using IL-15 and feeder line co-culture. We assessed global transcriptional profile, specific activating and inhibitory receptors of NK function, and principal component analysis (PCA) for variation between treatment groups (FDR &amp;lt;0.05). Results: Of 8 dogs, 2 demonstrated &amp;gt; 100-day survival with 1 stable disease and 1 partial response based on RECIST criteria. DGE revealed distinct transcriptional profiles between the ex vivo resting, IL-15 and co-cultured canine NK cells. Among treated patients, hierarchical clustering revealed that in vivo NK cell transcriptional signatures grouped by individual dog, and not amount of time exposed to treatment. PCA showed in vivo profiles of the long term survivors were distinctly separate from the non-responding patients (PC1 38%, PC2 12%). This suggests response to therapy could be determined by baseline NK cell characteristics rather than changes over time following treatment. Patient in vivo NK cell transcription profiles most closely resembled those of ex vivo resting NK cells and not IL-15 or co-culture activated (PC1 43%, PC2 19%), likely reflecting key differences in activation ex vivo vs in vivo. Key genes induced in vivo (&amp;gt;20X) post inhalation of rhIL-15 include DLA-DRA, B2M, and thymosin beta 4, while key genes induced ex vivo post rhIL-15 exposure include CD96, KLRB1, and SPP1/OPN. Conclusion: In this first transcriptomic sequencing of dog NK cells, we demonstrate distinct gene profiles of ex vivo activated NK cells from healthy donors compared to circulating NK cells from dogs receiving inhaled rhIL-15 on clinical trial. Baseline NK cell profiles appear to predict response more than changes over time. These data highlight the strength of the outbred dog model in speeding novel immunotherapy and biomarker studies. Citation Format: Alicia A. Gingrich, Taylor Reiter, Sean Judge, Daniel York, Mio Yanagisawa, Ian Sturgill, Rachel Brady, Kevin Stoffel, Arta Monjazeb, C. Titus Brown, Robert Rebhun, Robert J. Canter. Transcriptomic profiles improve characterization natural killer cells and predict response to cytokine therapy in clinical trial for dogs with pulmonary metastases [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1339.

Современный подход в иммунотерапии рака

В работе рассматриваются основные направления и результаты экспериментальных и клинических исследований иммунотерапии рака. Обсуждаются перспективы использования и реализации онколитического потенциала вирусов. Опираясь на результаты собственных исследований и анализ литературных данных, появилась необходимость всесторонней оценки возможностей применения онколитических вирусов в терапии пациентов с онкопатологией.

Leveraging Patient-Derived Models for Immunotherapy Research.

As cancer immunotherapies become mainstream for the treatment of many different cancer types and the numbers of new agents continue to increase, the need for experimental models is also rising. An approach to develop and study models for immune-oncology that has garnered intense interest in recent years is that of using patient-derived models. Patient-derived models can recapitulate many of the features and heterogeneity of the corresponding human tumors. Historically these models have been used to study cancer cell-intrinsic properties of tumors and drugs that target tumor cells directly. In recent years, increasing recognition of the role immune cells play in cancer and how these represent good therapeutic targets has led to efforts to optimize and use patient-derived models for cancer immunotherapy studies. Patient-derived models are now being used to study the properties of cancer cells that modulate their ability to respond to immune stimulation. Further efforts are underway to use and develop patient-derived models that incorporate human immune cells in vitro and in vivo (humanized mice) so that cancer cell-immune cell interactions can be studied in the context of cancer immunotherapies. As these models are further refined, leveraging patient-derived models for cancer immunotherapy research can provide insight into mechanisms of sensitivity and resistance to cancer immunotherapies, uncover new targets, reveal how specific agents work, and be used to evaluate the antitumor efficacy of therapeutic regimens.

Abstract B78: Co-potentiation of human T cells to identify subdominant tumor neoantigens from melanoma patients responding to immune checkpoint blockade

Abstract Malignant melanoma is the most aggressive form of skin cancer, accounting for 10,000 deaths annually. Largely resistant to conventional cytotoxic chemotherapy and radiation, immune checkpoint blockade (ICB) therapies have revolutionized patient care, accounting for partial or complete responses in up to 70% of patients. An important aspect of cancer elimination is activation of the cytotoxic T lymphocyte (CTL) response. Recent data suggest that ICB mediated broadening of the peripheral blood CTL repertoire correlates with good clinical outcomes. Thus, recent studies in cancer immunotherapy have focused on targeting tumor neoantigens (tNeoAg) with cancer vaccines for use in combination with ICB. Computational algorithm-driven predictions of immunogenic tNeoAgs yield vast numbers of potential peptide targets, only a fraction of which may be immunogenic in patients. This likely contributed to the modest success of cancer vaccines targeting predicted, high-avidity tNeoAgs, emphasizing the importance of identifying true tumor rejection antigens, including sub-dominant antigens, outside the scope of predictive models. In addition to promoting T cell-mediated tumor rejection, ICB often comes at the expense of treatment-induced immune-related adverse effects (irAE) that frequently require discontinuation of treatment. Modulation of ICB towards antitumor immunity and away from autoimmunity may dramatically improve the therapeutic index of modern cancer therapy. This appears feasible considering the clinical observation that antitumor efficacy does not correlate with type/severity of irAE, suggesting that the mechanisms of both processes, though likely related (CTL mediated), may not be identical. We aim to identify and separate non-cross-reacting antigenic targets mediating tumor rejection from those mediating irAEs to enable therapeutic interventions that maximize the efficacy of ICB, expanding tumor-specific CTLs with vaccines while minimizing irAEs through desensitization. We have access to samples from responding/nonresponding melanoma patients subjected to ICB with varying degrees of irAEs. We have designed an experimental approach that combines established mass spectrometry and sequencing techniques to identify peptides and matching TCR clones with a novel strategy that targets the TCR-associated CD3 complex to allow inclusion of subdominant antigens in our studies. T-cell “co-potentiation” is achieved when anti-CD3 monovalent Fabs induce a conformational change in the CD3 complex that sustains the T-cell response to weak antigenic stimulation. We have successfully used anti-human CD3 Fabs to co-potentiate in vitro the activation and subsequent response to weak peptide-HLA/TCR interactions of human T cells found in PBMCs isolated from healthy donors. Our preliminary data suggest T-cell co-potentiation may allow identification of ICB-induced CTL clones specific for subdominant tNeoAgs and irAE targets in patients with melanoma undergoing active immunotherapy. Citation Format: Laura Elsbernd, Alfreda Nelson, Hien Huynh, Michele Hoffmann, Christopher Parks, Wendy Nevala, Shari Sutor, Larry Pease, Adam Schrum, Diana Gil, Svetomir N. Markovic. Co-potentiation of human T cells to identify subdominant tumor neoantigens from melanoma patients responding to immune checkpoint blockade [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B78.

Biological and therapeutic advances in the pursuit of effective immunotherapy for prostate cancer.

The successful development of effective cancer immunotherapy, in particular immune checkpoint inhibitors, has changed the treatment paradigm of many tumor types. In light of the limited efficacy of checkpoint inhibitors demonstrated in recent clinical trials in refractory prostate cancer, this review highlights important recent and ongoing studies that are shaping the pursuit of effective immunotherapy for prostate cancer. We review two overarching themes with respect to recent studies of prostate cancer immunotherapy: evolving therapeutic strategies and novel biological findings, including the landscape of predictive biomarkers of immunotherapy response. Novel and combinatorial immunotherapy strategies are being implemented across the clinical spectrum of prostate cancer. Greater understanding of complex tumor-immune interactions and the determinants of therapy response in prostate cancer is an area of intense investigation, and will inform both translational and clinical immuno-oncology research in the field.

TMOD-07. IN SITU MICROSCOPY OF DRUG AND IMMUNE CELL INTERACTIONS AGAINST BIOFABRICATED TUMOR MODELS

Abstract BACKGROUND &amp; SIGNIFICANCE Cancer is a disease in 3-dimensions and there is a desperate need for infrastructure and models to study immuno-oncology treatment strategies in 3D. A new culture system for long-duration maturation of patient derived microtumors has been developed. This system enables in situconfocal imaging and movies of T-cells and tumor model interactions, including measurements of T-cell migration, infiltration, and killing. HYPOTHESIS: Biofabrication of 3D microtumors using bioprinting in transparent soft granular gel media can facilitate the precise arrangement and stability of extra-cellular matrix components, tumor, and immune cells while maintaining continuous liquid perfusion. METHODS The design, development, and validation of a modular negative pressure perfusion system controls the transport of liquid growth medium, drugs, antibodies, growth factors, and metabolic waste management in 3D. In situ confocal fluorescent microscopy measures cell positions, velocities, and viability during experiments. Steady perfusion velocities are controlled through negative pressure, and velocities from 1–100 nm/s can be achieved. RESULTS Cell motility, adhesion, and dynamic rearrangement of fibroblasts and endothelial cells within a 3D co-culture of microtumors evolved dramatically over the first 72 hours. Tracking of activated CD8+ T-cells revealed super-diffusive motion in the presence of 3D tumors with a range of 250 µm. Activated T-cell migration speeds have been measured to be between 1.3–2.0 um/min in the 3D media, and preliminary estimates of T cell migration forces are on the order of 1 nN. Arrival of CD8+ T-cells to the tumors within the first 30 minutes revealed cell killing which continued for over 3-hours and resulted in a 2-fold reduction in tumor cell numbers. CONCLUSIONS This integrated system of 3D bioprinting, perfusion culture plates, and confocal microscopy enables in situ3D studies of cancer biology, immunotherapy, and drug treatment regimens and provides unique insights and measurements of immune cell invasion dynamics in 3D microtumors.

Cancer immunotherapy and flow cytometry in immunotherapy monitoring

Immunotherapy for cancer treatment continues to be developed and various strategies have been carried out including bioengineering. This endeavour requires development of technology, and efforts to find specific and sensitive tools to monitor immune responses during and after therapy. The purpose of this mini-review was to discuss cancer immunotherapy using T cell and immune checkpoint blockade therapy, as well as immunotherapy monitoring methods using flow cytometry (FCM). Bioengineering of T lymphocytes for immunotherapy and immune checkpoint blockades can be combined with nanoparticles as drug delivery carrier against cancer to increase drug distribution to tumor cells, as well as T cell stimulation regulation to reduce autoimmune effects. In addition, T cell engineering can also prevent Host versus Graft alloreactivity in chimeric antigen receptor (CAR) T cell administration. FCM is a monitoring method that is widely used in pre-clinical and clinical cancer immunotherapy studies.