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Graded index (GRIN) lens endoscopy has broadly benefited biomedical microscopic imaging by enabling accessibility to sites not reachable by traditional benchtop microscopes. It is a long-held notion that GRIN lenses can only be used as rigid probes, which may limit their potential for certain applications. Here, we describe bendable and long-range GRIN microimaging probes for a variety of potential micro-endoscopic biomedical applications. Using a two-photon fluorescence imaging system, we have experimentally demonstrated the feasibility of three-dimensional imaging through a 500-µm-diameter and ∼11 cm long GRIN lens subject to a cantilever beam-like deflection with a minimum bend radius of ∼25 cm. Bend-induced perturbation to the field of view and resolution has also been investigated quantitatively. Our development alters the conventional notion of GRIN lenses and enables a range of innovative applications. For example, the demonstrated flexibility is highly desirable for implementation into current and emerging minimally invasive clinical procedures, including a pioneering microdevice for high-throughput cancer drug selection.

Systematically identifying synergistic combinations of targeted agents and immunotherapies for cancer treatments remains difficult. In this study, we integrated high-throughput and high-content techniques—an implantable microdevice to administer multiple drugs into different sites in tumors at nanodoses and multiplexed imaging of tumor microenvironmental states—to investigate the tumor cell and immunological response signatures to different treatment regimens. Using a mouse model of breast cancer, we identified effective combinations from among numerous agents within days. In vivo studies in three immunocompetent mammary carcinoma models demonstrated that the predicted combinations synergistically increased therapeutic efficacy. We identified at least five promising treatment strategies, of which the panobinostat, venetoclax and anti-CD40 triple therapy was the most effective in inducing complete tumor remission across models. Successful drug combinations increased spatial association of cancer stem cells with dendritic cells during immunogenic cell death, suggesting this as an important mechanism of action in long-term breast cancer control.

Tumor-infiltrating immune cells experience significant metabolic reprogramming in the tumor microenvironment (TME), and they share similar metabolic pathways and nutrient needs with malignant cells. This positions these cell types in direct nutrient competition in the TME. We currently lack a complete understanding of the similarities, differences, and functional consequences of the metabolic pathways utilized by activated immune cells from different lineages

Systematically identifying synergistic combinations between targeted agents and immunotherapies in cancer based on genomic or other static biomarkers remains elusive. Here we integrate two novel high-content and high-throughput techniques, an implantable microdevice to administer multiple drugs into different sites in tumors at nanodoses; and spatial systems analysis of tumor microenvironmental states to describe tumor cell and immunological response signatures and rapidly, within days, identify effective combinations from among numerous agents. We demonstrate in systemic follow-up studies across three mammary carcinoma models that combinations identified by this approach lead to highly synergistic effects. Biomarkers associated with resistance to each agent allowed us to prioritize at least five novel treatment strategies of which the panobinostat/venetoclax/anti-CD40 was the most effective inducing complete tumor control across models. We show that spatial association of cancer stem cells with dendritic cells during immunogenic cell death is a potential mechanism of action underlying long-term breast cancer control.

Systematically identifying synergistic combinations of targeted agents and immunotherapies for cancer treatments remains difficult. In this study, we integrated high-throughput and high-content techniques-an implantable microdevice to administer multiple drugs into different sites in tumors at nanodoses and multiplexed imaging of tumor microenvironmental states-to investigate the tumor cell and immunological response signatures to different treatment regimens. Using a mouse model of breast cancer, we identified effective combinations from among numerous agents within days. In vivo studies in three immunocompetent mammary carcinoma models demonstrated that the predicted combinations synergistically increased therapeutic efficacy. We identified at least five promising treatment strategies, of which the panobinostat, venetoclax and anti-CD40 triple therapy was the most effective in inducing complete tumor remission across models. Successful drug combinations increased spatial association of cancer stem cells with dendritic cells during immunogenic cell death, suggesting this as an important mechanism of action in long-term breast cancer control.

BackgroundIdentifying how to optimally combine immunotherapies with other available anti-cancer therapies is a major challenge in oncology. A systematic method to screen many potential combination therapies ideally in vivo has remained elusive. We have utilized an implantable microdevice (IMD) performing cassette microdosing that measures intratumor drug responses and anti-tumor immunity for 20 agents in parallel. For each of the agents, local tumor response is measured by cyclical immunofluorescence for deep cellular response phenotyping. This approach is combined with systemic administration of checkpoint inhibitors to examine whether local immunogenic cell death (ICD) induced by a given drug microdose potentiates the immunotherapy’s anti-tumor effect.MethodsThe measurements were performed in a humanized mouse model of renal cancer, patient derived xenograft (PDX) RXF488. The PDX is derived from a 68 year old male patient suffering from clear cell renal carcinoma. RXF488 was implanted subcutaneously in 30 NSG mice. Animals were stratified into 6 groups with n= 4–6. Humanization was performed by the intravenous injection of 5x10e6 human peripheral blood mononuclear cells (PBMC) prior to the first treatment. Systemic anti-PD1 treatment was applied in the presence and absence of the microdevice loaded with eleven different drugs. Control groups received the microdevice in the presence or absence of PBMC. Beside the histological examination of the tumor tissue, flow cytometry (FC) was performed on bone marrow, spleen and tumor tissue to determine infiltration of human immune cells.ResultsFC analyses revealed no influence of the treatment on the human immune cells in bone marrow and spleen. The anti-PD1 treatment induced an increase in huCD45+ cells specifically in the tumor tissue and a decrease of the CD4/CD8 ratio in these cells only 48h after treatment. Our combination screen identified LXH254, Sorafenib and Doxorubicin exhibiting the highest increase in apoptosis induction when combined with checkpoint inhibitors. The increased efficacy from immunotherapy administration coincided with increased induction of ICD. We were able to verify the results of the screening experiment in a conventional setting with systemic combination treatment in the same PDX model.ConclusionsOur results demonstrate that local tumor response signatures of ICD can be used to systemically identify synergistic combinations of a range of drugs with immunotherapy on a tumor specific basis. The approach may represent a new paradigm for efficient in vivo screening of novel combinations, particularly with combinations involving immunotherapies.

SUMMARYBetter methods are needed to identify effective combinations of immunotherapies with chemotherapies and targeted anti-cancer agents. Here we present a Multiplex Implantable Microdevice Assay (MIMA) system for rapid in vivo assessment of the effects of multiple, spatially separate anticancer drugs directly in the complex tumor microenvironment. In prototypic experiments, olaparib, lenvatinib, palbociclib, venetoclax, panobinostat, doxorubicin, and paclitaxel and combinations thereof were administered simultaneously to murine mammary tumor models. Quantitative multiplex immunohistochemistry and spatial systems analyses of each local drug response defined cellular relations of fibroblasts, endothelial cells, immune lineages, immunogenic cell death, tumor proliferation and/or cancer stem cells that were used to predict effective drug combinations. A predicted combination of panobinostat, venetoclax and anti-CD40 showed long-term anti-tumor efficacy in multiple mouse models with no observable toxicity when administered systemically. Future MIMA use promises to design effective drug combinations for tumor cell control and immune activation on a personalized basis.

Background: Tumor heterogeneity is increasingly being recognized as a major source of variability in the histopathological assessment of drug responses. Quantitative analysis of immunohistochemistry (IHC) and immunofluorescence (IF) images using biomarkers that capture spatialpatterns of distinct tumor biology and drug concentration in tumors is of high interest to the field. Methods: We have developed an image analysis pipeline to measure drug response using IF and IHC images along spatial gradients of local drug release from a tumor-implantable drug delivery microdevice. The pipeline utilizes a series of user-interactive python scripts and CellProfiler pipelines with custom modules to perform image and spatial analysis of regions of interest within whole-slide images. Results: Worked examples demonstrate that intratumor measurements such as apoptosis, cell proliferation, and immune cell population density can be quantitated in a spatially and drug concentration-dependent manner, establishing in vivo profiles of pharmacodynamics and pharmacokinetics in tumors. Conclusions: Spatial image analysis of tumor response along gradients of local drug release is achievable in high throughput. The major advantage of this approach is the use of spatially aware annotation tools to correlate drug gradients with drug effects in tumors in vivo.

Advances in the intratumor measurement of drug responses have included a pioneering biomedical microdevice for high throughput drug screening in vivo, which was further advanced by integrating a graded-index lens based two-dimensional fluorescence micro-endoscope to monitor tissue responses in situ across time. While the previous system provided a bulk measurement of both drug delivery and tissue response from a given region of the tumor, it was incapable of visualizing drug distribution and tissue responses in a three-dimensional (3D) way, thus missing the critical relationship between drug concentration and effect. Here we demonstrate a next-generation system that couples multiplexed intratumor drug release with continuous 3D spatial imaging of the tumor microenvironment via the integration of a miniaturized two-photon micro-endoscope. This enables optical sectioning within the live tissue microenvironment to effectively profile the entire tumor region adjacent to the microdevice across time. Using this novel microimaging-microdevice (MI-MD) system, we successfully demonstrated the four-dimensional imaging (3 spatial dimensions plus time) of local drug delivery in tissue phantom and tumors. Future studies include the use of the MI-MD system for monitoring of localized intra-tissue drug release and concurrent measurement of tissue responses in live organisms, with applications to study drug resistance due to nonuniform drug distribution in tumors, or immune cell responses to anti-cancer agents.

Patient-specific dose response against chemotherapeutics can be assessed through local in situ release of drugs at sub-therapeutic concentrations. Such controlled release can be performed in patient-derived xenografts (PDXs), which offer pre-clinical methods for mimicking the tumor microenvironment. However, the prolonged co-existence of intermingled human and mouse tissues poses a number of challenges for histological image analysis. Manual annotation of regions of human tissue is labor-intensive and lacks reproducibility and scalability, complicating the investigation of multiplexed local drug effects near drug-dispensing microdevices. To this end, we apply a random forest algorithm for segmenting histological images to obtain binary masks for refined image analysis. Region-of-interest masks obtained using this supervised learning approach allow for a spatially refined assessment of the dose response in heterogeneous tissue8. We achieved a Dice similarity coefficient score (DSC) of 0.56 with the random forest classifier.

By observing the activity of anti-cancer agents directly in tumors, there is potential to greatly expand our understanding of drug response and develop more personalized cancer treatments. Implantable microdevices (IMD) have been recently developed to deliver microdoses of chemotherapeutic agents locally into confined regions of live tumors, the tissue can be subsequently removed and analyzed to evaluate drug response. This method has the potential to rapidly screen multiple drugs, but requires surgical tissue removal and only evaluates drug response at a single timepoint when the tissue is excised. Here, we describe a “lab-in-a-tumor” implantable microdevice (LIT-IMD) platform to image cell-death drug response within a live tumor, without requiring surgical resection or tissue processing. The LIT-IMD is inserted into a live tumor and delivers multiple drug microdoses into spatially discrete locations. In parallel, it locally delivers microdose levels of a fluorescent cell-death assay, which diffuses into drug-exposed tissues and accumulates at sites of cell death. An integrated miniaturized fluorescence imaging probe images each region to evaluate drug-induced cell death. We demonstrate ability to evaluate multi-drug response over 8 h using murine tumor models and show correlation with gold-standard conventional fluorescence microscopy and histopathology. This is the first demonstration of a fully integrated platform for evaluating multiple chemotherapy responses in situ. This approach could enable a more complete understanding of drug activity in live tumors, and could expand the utility of drug-response measurements to a wide range of settings where surgery is not feasible.