DeepCEL0 for 2D single-molecule localization in fluorescence microscopy.

MOTIVATION: In fluorescence microscopy, single-molecule localization microscopy (SMLM) techniques aim at localizing with high-precision high-density fluorescent molecules by stochastically activating and imaging small subsets of blinking emitters. Super resolution plays an important role in this field since it allows to go beyond the intrinsic light diffraction limit. RESULTS: In this work, we propose a deep learning-based algorithm for precise molecule localization of high-density frames acquired by SMLM techniques whose ℓ2-based loss function is regularized by non-negative and ℓ0-based constraints. The ℓ0 is relaxed through its continuous exact ℓ0 (CEL0) counterpart. The arising approach, named DeepCEL0, is parameter-free, more flexible, faster and provides more precise molecule localization maps if compared to the other state-of-the-art methods. We validate our approach on both simulated and real fluorescence microscopy data. AVAILABILITY AND IMPLEMENTATION: DeepCEL0 code is freely accessible at https://github.com/sedaboni/DeepCEL0.

Apoptosis mapping in space and time of 3D tumor ecosystems reveals transmissibility of cytotoxic cancer death.

The emerging tumor-on-chip (ToC) approaches allow to address biomedical questions out of reach with classical cell culture techniques: in biomimetic 3D hydrogels they partially reconstitute ex vivo the complexity of the tumor microenvironment and the cellular dynamics involving multiple cell types (cancer cells, immune cells, fibroblasts, etc.). However, a clear bottleneck is the extraction and interpretation of the rich biological information contained, sometime hidden, in the cell co-culture videos. In this work, we develop and apply novel video analysis algorithms to automatically measure the cytotoxic effects on human cancer cells (lung and breast) induced either by doxorubicin chemotherapy drug or by autologous tumor-infiltrating cytotoxic T lymphocytes (CTL). A live fluorescent dye (red) is used to selectively pre-stain the cancer cells before co-cultures and a live fluorescent reporter for caspase activity (green) is used to monitor apoptotic cell death. The here described open-source computational method, named STAMP (spatiotemporal apoptosis mapper), extracts the temporal kinetics and the spatial maps of cancer death, by localizing and tracking cancer cells in the red channel, and by counting the red to green transition signals, over 2-3 days. The robustness and versatility of the method is demonstrated by its application to different cell models and co-culture combinations. Noteworthy, this approach reveals the strong contribution of primary cancer-associated fibroblasts (CAFs) to breast cancer chemo-resistance, proving to be a powerful strategy to investigate intercellular cross-talks and drug resistance mechanisms. Moreover, we defined a new parameter, the 'potential of death induction', which is computed in time and in space to quantify the impact of dying cells on neighbor cells. We found that, contrary to natural death, cancer death induced by chemotherapy or by CTL is transmissible, in that it promotes the death of nearby cancer cells, suggesting the release of diffusible factors which amplify the initial cytotoxic stimulus.

Polylactic is a Sustainable, Low Absorption, Low Autofluorescence Alternative to Other Plastics for Microfluidic and Organ-on-Chip Applications.

Organ-on-chip (OOC) devices are miniaturized devices replacing animal models in drug discovery and toxicology studies. The majority of OOC devices are made from polydimethylsiloxane (PDMS), an elastomer widely used in microfluidic prototyping, but posing a number of challenges to experimentalists, including leaching of uncured oligomers and uncontrolled absorption of small compounds. Here we assess the suitability of polylactic acid (PLA) as a replacement material to PDMS for microfluidic cell culture and OOC applications. We changed the wettability of PLA substrates and demonstrated the functionalization method to be stable over a time period of at least 9 months. We successfully cultured human cells on PLA substrates and devices, without coating. We demonstrated that PLA does not absorb small molecules, is transparent (92% transparency), and has low autofluorescence. As a proof of concept of its manufacturability, biocompatibility, and transparency, we performed a cell tracking experiment of prostate cancer cells in a PLA device for advanced cell culture.

A Camera Sensors-Based System to Study Drug Effects On In Vitro Motility: The Case of PC-3 Prostate Cancer Cells.

Cell motility is the brilliant result of cell status and its interaction with close environments. Its detection is now possible, thanks to the synergy of high-resolution camera sensors, time-lapse microscopy devices, and dedicated software tools for video and data analysis. In this scenario, we formulated a novel paradigm in which we considered the individual cells as a sort of sensitive element of a sensor, which exploits the camera as a transducer returning the movement of the cell as an output signal. In this way, cell movement allows us to retrieve information about the chemical composition of the close environment. To optimally exploit this information, in this work, we introduce a new setting, in which a cell trajectory is divided into sub-tracks, each one characterized by a specific motion kind. Hence, we considered all the sub-tracks of the single-cell trajectory as the signals of a virtual array of cell motility-based sensors. The kinematics of each sub-track is quantified and used for a classification task. To investigate the potential of the proposed approach, we have compared the achieved performances with those obtained by using a single-trajectory paradigm with the scope to evaluate the chemotherapy treatment effects on prostate cancer cells. Novel pattern recognition algorithms have been applied to the descriptors extracted at a sub-track level by implementing features, as well as samples selection (a good teacher learning approach) for model construction. The experimental results have put in evidence that the performances are higher when a further cluster majority role has been considered, by emulating a sort of sensor fusion procedure. All of these results highlighted the high strength of the proposed approach, and straightforwardly prefigure its use in lab-on-chip or organ-on-chip applications, where the cell motility analysis can be massively applied using time-lapse microscopy images.

Online Feature Selection for Robust Classification of the Microbiological Quality of Traditional Vanilla Cream by Means of Multispectral Imaging.

The performance of an Unsupervised Online feature Selection (UOS) algorithm was investigated for the selection of training features of multispectral images acquired from a dairy product (vanilla cream) stored under isothermal conditions. The selected features were further used as input in a support vector machine (SVM) model with linear kernel for the determination of the microbiological quality of vanilla cream. Model training (n = 65) was based on two batches of cream samples provided directly by the manufacturer and stored at different isothermal conditions (4, 8, 12, and 15 °C), whereas model testing (n = 132) and validation (n = 48) were based on real life conditions by analyzing samples from different retail outlets as well as expired samples from the market. Qualitative analysis was performed for the discrimination of cream samples in two microbiological quality classes based on the values of total viable counts [TVC ≤ 2.0 log CFU/g (fresh samples) and TVC ≥ 6.0 log CFU/g (spoiled samples)]. Results exhibited good performance with an overall accuracy of classification for the two classes of 91.7% for model validation. Further on, the model was extended to include the samples in the TVC range 2-6 log CFU/g, using 1 log step to define the microbiological quality of classes in order to assess the potential of the model to estimate increasing microbial populations. Results demonstrated that high rates of correct classification could be obtained in the range of 2-5 log CFU/g, whereas the percentage of erroneous classification increased in the TVC class (5,6) that was close to the spoilage level of the product. Overall, the results of this study demonstrated that the UOS algorithm in tandem with spectral data acquired from multispectral imaging could be a promising method for real-time assessment of the microbiological quality of vanilla cream samples.

Integrating machine learning and biosensors in microfluidic devices: A review.

Microfluidic devices are increasingly widespread in the literature, being applied to numerous exciting applications, from chemical research to Point-of-Care devices, passing through drug development and clinical scenarios. Setting up these microenvironments, however, introduces the necessity of locally controlling the variables involved in the phenomena under investigation. For this reason, the literature has deeply explored the possibility of introducing sensing elements to investigate the physical quantities and the biochemical concentration inside microfluidic devices. Biosensors, particularly, are well known for their high accuracy, selectivity, and responsiveness. However, their signals could be challenging to interpret and must be carefully analysed to carry out the correct information. In addition, proper data analysis has been demonstrated even to increase biosensors' mentioned qualities. To this regard, machine learning algorithms are undoubtedly among the most suitable approaches to undertake this job, automatically learning from data and highlighting biosensor signals' characteristics at best. Interestingly, it was also demonstrated to benefit microfluidic devices themselves, in a new paradigm that the literature is starting to name "intelligent microfluidics", ideally closing this benefic interaction among these disciplines. This review aims to demonstrate the advantages of the triad paradigm microfluidics-biosensors-machine learning, which is still little used but has a great perspective. After briefly describing the single entities, the different sections will demonstrate the benefits of the dual interactions, highlighting the applications where the reviewed triad paradigm was employed.

Cell Electrokinetic Fingerprint: A Novel Approach Based on Optically Induced Dielectrophoresis (ODEP) for In-Flow Identification of Single Cells.

A novel optically induced dielectrophoresis (ODEP) system that can operate under flow conditions is designed for automatic trapping of cells and subsequent induction of 2D multi-frequency cell trajectories. Like in a "ping-pong" match, two virtual electrode barriers operate in an alternate mode with varying frequencies of the input voltage. The so-derived cell motions are characterized via time-lapse microscopy, cell tracking, and state-of-the-art machine learning algorithms, like the wavelet scattering transform (WST). As a cell-electrokinetic fingerprint, the dynamic of variation of the cell displacements happening, over time, is quantified in response to different frequency values of the induced electric field. When tested on two biological scenarios in the cancer domain, the proposed approach discriminates cellular dielectric phenotypes obtained, respectively, at different early phases of drug-induced apoptosis in prostate cancer (PC3) cells and for differential expression of the lectine-like oxidized low-density lipoprotein receptor-1 (LOX-1) transcript levels in human colorectal adenocarcinoma (DLD-1) cells. The results demonstrate increased discrimination of the proposed system and pose an additional basis for making ODEP-based assays addressing cancer heterogeneity for precision medicine and pharmacological research.

Assessing personalized responses to anti-PD-1 treatment using patient-derived lung tumor-on-chip.

There is a compelling need for approaches to predict the efficacy of immunotherapy drugs. Tumor-on-chip technology exploits microfluidics to generate 3D cell co-cultures embedded in hydrogels that recapitulate simplified tumor ecosystems. Here, we present the development and validation of lung tumor-on-chip platforms to quickly and precisely measure ex vivo the effects of immune checkpoint inhibitors on T cell-mediated cancer cell death by exploiting the power of live imaging and advanced image analysis algorithms. The integration of autologous immunosuppressive FAP+ cancer-associated fibroblasts impaired the response to anti-PD-1, indicating that tumors-on-chips are capable of recapitulating stroma-dependent mechanisms of immunotherapy resistance. For a small cohort of non-small cell lung cancer patients, we generated personalized tumors-on-chips with their autologous primary cells isolated from fresh tumor samples, and we measured the responses to anti-PD-1 treatment. These results support the power of tumor-on-chip technology in immuno-oncology research and open a path to future clinical validations.

Extracting fuzzy classification rules from texture segmented HRCT lung images.

Automatic tools for detection and identification of lung and lesion from high-resolution CT (HRCT) are becoming increasingly important both for diagnosis and for delivering high-precision radiation therapy. However, development of robust and interpretable classifiers still presents a challenge especially in case of non-small cell lung carcinoma (NSCLC) patients. In this paper, we have attempted to devise such a classifier by extracting fuzzy rules from texture segmented regions from HRCT images of NSCLC patients. A fuzzy inference system (FIS) has been constructed starting from a feature extraction procedure applied on overlapping regions from the same organs and deriving simple if-then rules so that more linguistically interpretable decisions can be implemented. The proposed method has been tested on 138 regions extracted from CT scan images acquired from patients with lung cancer. Assuming two classes of tissues C1 (healthy tissues) and C2 (lesion) as negative and positive, respectively; preliminary results report an AUC = 0.98 for lesions and AUC = 0.93 for healthy tissue, with an optimal operating condition related to sensitivity = 0.96, and specificity = 0.98 for lesions and sensitivity 0.99, and specificity = 0.94 for healthy tissue. Finally, the following results have been obtained: false-negative rate (FNR) = 6 % (C1), FNR = 2 % (C2), false-positive rate (FPR) = 4 % (C1), FPR = 3 % (C2), true-positive rate (TPR) = 94 %, (C1) and TPR = 98 % (C2).

Automatic detection of the nipple in screen-film and full-field digital mammograms using a novel Hessian-based method.

Automatic detection of the nipple in mammograms is an important step in computerized systems that combine multiview information for accurate detection and diagnosis of breast cancer. Locating the nipple is a difficult task owing to variations in image quality, presence of noise, and distortion and displacement of the breast tissue due to compression. In this work, we propose a novel Hessian-based method to locate automatically the nipple in screen-film and full-field digital mammograms (FFDMs). The method includes detection of a plausible nipple/retroareolar area in a mammogram using geometrical constraints, analysis of the gradient vector field by mean and Gaussian curvature measurements, and local shape-based conditions. The proposed procedure was tested on 566 mammographic images consisting of 372 randomly selected scanned films from two public databases (mini-MIAS and DDSM), and 194 digital mammograms acquired with a GE Senographe 2000D FFDM system. A radiologist independently marked the centers of the nipples for evaluation of the results. The average error obtained was 6.7 mm (22 pixels) with reference to the center of the nipple as identified by the radiologist. Only two out of the 566 detected nipples (0.35 %) had an error larger than 50 mm. The method was also directly compared with two other techniques for the detection of the nipple. The results indicate that the proposed method outperforms other algorithms presented in the literature and can be used to identify accurately the nipple on various types of mammographic images.

Electro-Optical Classification of Pollen Grains via Microfluidics and Machine Learning.

OBJECTIVE: In aerobiological monitoring and agriculture there is a pressing need for accurate, label-free and automated analysis of pollen grains, in order to reduce the cost, workload and possible errors associated to traditional approaches. METHODS: We propose a new multimodal approach that combines electrical sensing and optical imaging to classify pollen grains flowing in a microfluidic chip at a throughput of 150 grains per second. Electrical signals and synchronized optical images are processed by two independent machine learning-based classifiers, whose predictions are then combined to provide the final classification outcome. RESULTS: The applicability of the method is demonstrated in a proof-of-concept classification experiment involving eight pollen classes from different taxa. The average balanced accuracy is 78.7% for the electrical classifier, 76.7% for the optical classifier and 84.2% for the multimodal classifier. The accuracy is 82.8% for the electrical classifier, 84.1% for the optical classifier and 88.3% for the multimodal classifier. CONCLUSION: The multimodal approach provides better classification results with respect to the analysis based on electrical or optical features alone. SIGNIFICANCE: The proposed methodology paves the way for automated multimodal palynology. Moreover, it can be extended to other fields, such as diagnostics and cell therapy, where it could be used for label-free identification of cell populations in heterogeneous samples.

Direct imaging and automatic analysis in tumor-on-chip reveal cooperative antitumoral activity of immune cells and oncolytic vaccinia virus.

Organ-on-chip and tumor-on-chip microfluidic cell cultures represent a fast-growing research field for modelling organ functions and diseases, for drug development, and for promising applications in personalized medicine. Still, one of the bottlenecks of this technology is the analysis of the huge amount of bio-images acquired in these dynamic 3D microenvironments, a task that we propose to achieve by exploiting the interdisciplinary contributions of computer science and electronic engineering. In this work, we apply this strategy to the study of oncolytic vaccinia virus (OVV), an emerging agent in cancer immunotherapy. Infection and killing of cancer cells by OVV were recapitulated and directly imaged in tumor-on-chip. By developing and applying appropriate image analysis strategies and advanced automatic algorithms, we uncovered synergistic cooperation of OVV and immune cells to kill cancer cells. Moreover, we observed that the kinetics of immune cells were modified in presence of OVV and that these immune modulations varied during the course of infection. A correlation between cancer cell infection and cancer-immune interaction time was pointed out, strongly supporting a cause-effect relationship between infection of cancer cells and their recognition by the immune cells. These results shed new light on the mode of action of OVV, and suggest new clinical avenues for immunotherapy developments.

Oncoimmunology Meets Organs-on-Chip.

Oncoimmunology represents a biomedical research discipline coined to study the roles of immune system in cancer progression with the aim of discovering novel strategies to arm it against the malignancy. Infiltration of immune cells within the tumor microenvironment is an early event that results in the establishment of a dynamic cross-talk. Here, immune cells sense antigenic cues to mount a specific anti-tumor response while cancer cells emanate inhibitory signals to dampen it. Animals models have led to giant steps in this research context, and several tools to investigate the effect of immune infiltration in the tumor microenvironment are currently available. However, the use of animals represents a challenge due to ethical issues and long duration of experiments. Organs-on-chip are innovative tools not only to study how cells derived from different organs interact with each other, but also to investigate on the crosstalk between immune cells and different types of cancer cells. In this review, we describe the state-of-the-art of microfluidics and the impact of OOC in the field of oncoimmunology underlining the importance of this system in the advancements on the complexity of tumor microenvironment.

Recursive Deep Prior Video: A super resolution algorithm for time-lapse microscopy of organ-on-chip experiments.

Biological experiments based on organ-on-chips (OOCs) exploit light Time-Lapse Microscopy (TLM) for a direct observation of cell movement that is an observable signature of underlying biological processes. A high spatial resolution is essential to capture cell dynamics and interactions from recorded experiments by TLM. Unfortunately, due to physical and cost limitations, acquiring high resolution videos is not always possible. To overcome the problem, we present here a new deep learning-based algorithm that extends the well-known Deep Image Prior (DIP) to TLM Video Super Resolution without requiring any training. The proposed Recursive Deep Prior Video method introduces some novelties. The weights of the DIP network architecture are initialized for each of the frames according to a new recursive updating rule combined with an efficient early stopping criterion. Moreover, the DIP loss function is penalized by two different Total Variation-based terms. The method has been validated on synthetic, i.e., artificially generated, as well as real videos from OOC experiments related to tumor-immune interaction. The achieved results are compared with several state-of-the-art trained deep learning Super Resolution algorithms showing outstanding performances.

Early Prediction of Breast Cancer Recurrence for Patients Treated with Neoadjuvant Chemotherapy: A Transfer Learning Approach on DCE-MRIs.

Cancer treatment planning benefits from an accurate early prediction of the treatment efficacy. The goal of this study is to give an early prediction of three-year Breast Cancer Recurrence (BCR) for patients who underwent neoadjuvant chemotherapy. We addressed the task from a new perspective based on transfer learning applied to pre-treatment and early-treatment DCE-MRI scans. Firstly, low-level features were automatically extracted from MR images using a pre-trained Convolutional Neural Network (CNN) architecture without human intervention. Subsequently, the prediction model was built with an optimal subset of CNN features and evaluated on two sets of patients from I-SPY1 TRIAL and BREAST-MRI-NACT-Pilot public databases: a fine-tuning dataset (70 not recurrent and 26 recurrent cases), which was primarily used to find the optimal subset of CNN features, and an independent test (45 not recurrent and 17 recurrent cases), whose patients had not been involved in the feature selection process. The best results were achieved when the optimal CNN features were augmented by four clinical variables (age, ER, PgR, HER2+), reaching an accuracy of 91.7% and 85.2%, a sensitivity of 80.8% and 84.6%, a specificity of 95.7% and 85.4%, and an AUC value of 0.93 and 0.83 on the fine-tuning dataset and the independent test, respectively. Finally, the CNN features extracted from pre-treatment and early-treatment exams were revealed to be strong predictors of BCR.

NeuriTES. Monitoring neurite changes through transfer entropy and semantic segmentation in bright-field time-lapse microscopy.

One of the most challenging frontiers in biological systems understanding is fluorescent label-free imaging. We present here the NeuriTES platform that revisits the standard paradigms of video analysis to detect unlabeled objects and adapt to the dynamic evolution of the phenomenon under observation. Object segmentation is reformulated using robust algorithms to assure regular cell detection and transfer entropy measures are used to study the inter-relationship among the parameters related to the evolving system. We applied the NeuriTES platform to the automatic analysis of neurites degeneration in presence of amyotrophic lateral sclerosis (ALS) and to the study of the effects of a chemotherapy drug on living prostate cancer cells (PC3) cultures. Control cells have been considered in both the two cases study. Accuracy values of 93% and of 92% are achieved, respectively. NeuriTES not only represents a tool for investigation in fluorescent label-free images but demonstrates to be adaptable to individual needs.

Early prediction of neoadjuvant chemotherapy response by exploiting a transfer learning approach on breast DCE-MRIs.

The dynamic contrast-enhanced MR imaging plays a crucial role in evaluating the effectiveness of neoadjuvant chemotherapy (NAC) even since its early stage through the prediction of the final pathological complete response (pCR). In this study, we proposed a transfer learning approach to predict if a patient achieved pCR (pCR) or did not (non-pCR) by exploiting, separately or in combination, pre-treatment and early-treatment exams from I-SPY1 TRIAL public database. First, low-level features, i.e., related to local structure of the image, were automatically extracted by a pre-trained convolutional neural network (CNN) overcoming manual feature extraction. Next, an optimal set of most stable features was detected and then used to design an SVM classifier. A first subset of patients, called fine-tuning dataset (30 pCR; 78 non-pCR), was used to perform the optimal choice of features. A second subset not involved in the feature selection process was employed as an independent test (7 pCR; 19 non-pCR) to validate the model. By combining the optimal features extracted from both pre-treatment and early-treatment exams with some clinical features, i.e., ER, PgR, HER2 and molecular subtype, an accuracy of 91.4% and 92.3%, and an AUC value of 0.93 and 0.90, were returned on the fine-tuning dataset and the independent test, respectively. Overall, the low-level CNN features have an important role in the early evaluation of the NAC efficacy by predicting pCR. The proposed model represents a first effort towards the development of a clinical support tool for an early prediction of pCR to NAC.

Combining microfluidics with machine learning algorithms for RBC classification in rare hereditary hemolytic anemia.

Combining microfluidics technology with machine learning represents an innovative approach to conduct massive quantitative cell behavior study and implement smart decision-making systems in support of clinical diagnostics. The spleen plays a key-role in rare hereditary hemolytic anemia (RHHA), being the organ responsible for the premature removal of defective red blood cells (RBCs). The goal is to adapt the physiological spleen filtering strategy for in vitro study and monitoring of blood diseases through RBCs shape analysis. Then, a microfluidic device mimicking the slits of the spleen red pulp area and video data analysis are combined for the characterization of RBCs in RHHA. This microfluidic unit is designed to evaluate RBC deformability by maintaining them fixed in planar orientation, allowing the visual inspection of RBC's capacity to restore their original shape after crossing microconstrictions. Then, two cooperative learning approaches are used for the analysis: the majority voting scheme, in which the most voted label for all the cell images is the class assigned to the entire video; and the maximum sum of scores to decide the maximally scored class to assign. The proposed platform shows the capability to discriminate healthy controls and patients with an average efficiency of 91%, but also to distinguish between RHHA subtypes, with an efficiency of 82%.

Lateral Acromioplasty has a Positive Impact on Rotator Cuff Repair in Patients with a Critical Shoulder Angle Greater than 35 Degrees.

BACKGROUND: A Critical Shoulder Angle (CSA), evaluated on plain radiographs, greater than 35° is considered predictive of rotator cuff tears. The present prospective comparative study aimed, firstly, to develop a formula to calculate the amount of acromion that should be resected performing a lateral acromioplasty and, secondly, verify whether lateral acromioplasty to reduce the CSA associated with arthroscopic cuff repair decreased the rate of recurrence of the tears, and impacted favorably on clinical postoperative outcomes. METHODS: Patients undergoing arthroscopic rotator cuff repair (RCR) for rotator cuff tears with a CSA greater than 35° were included in this study and divided into two groups, based on whether the CSA had been reduced by arthroscopic resection of the lateral portion of the acromion. A new mathematical formula was developed in order to quantify the amount of bone to be resected while performing the lateral acromioplasty. Patients with traumatic tears, previous surgery, osteoarthritis or plain radiographs, not classified as A1 according to Suter-Henninger, were excluded. Clinical and radiographic outcomes were assessed at a minimum of 2 years of follow-up considering the tear size. RESULTS: 289 patients were included in this study. Thirty-seven were lost to follow-up. Group A (Lateral acromioplasty) patients included: 38 small tears, 30 medium tears, 28 large tears and 22 massive tears; Group B (control group) was composed of 40 small tears, 30 medium tears, 30 large tears and 23 massive tears. The Constants Score value and retear Rate were, respectively, significant higher (p = 0.007 and p = 0.004) and lower (p = 0.029 and p = 0.028) in Group A, both in the Small-and Medium-size subgroups. No complications were outlined. The mediolateral width of the acromion was reduced, according to the preoperatively calculated measure. CONCLUSION: Arthroscopic lateral acromioplasty decreased the CSA within the favorable range (30°-35°) in all patients treated, resecting the amount of bone predicted by the mathematical formula. Lateral acromioplasty is a safe and reproducible technique which may prevent recurrence of rotator cuff tears in patients with small and medium lesions. LEVEL OF EVIDENCE: II.

High-throughput analysis of cell-cell crosstalk in ad hoc designed microfluidic chips for oncoimmunology applications.

Understanding the interactions between immune and cancer cells occurring within the tumor microenvironment is a prerequisite for successful and personalized anti-cancer therapies. Microfluidic devices, coupled to advanced microscopy systems and automated analytical tools, can represent an innovative approach for high-throughput investigations on immune cell-cancer interactions. In order to study such interactions and to evaluate how therapeutic agents can affect this crosstalk, we employed two ad hoc fabricated microfluidic platforms reproducing advanced 2D or 3D tumor immune microenvironments. In the first type of chip, we confronted the capacity of tumor cells embedded in Matrigel containing one drug or Matrigel containing a combination of two drugs to attract differentially immune cells, by fluorescence microscopy analyses. In the second chip, we investigated the migratory/interaction response of naïve immune cells to danger signals emanated from tumor cells treated with an immunogenic drug, by time-lapse microscopy and automated tracking analysis. We demonstrate that microfluidic platforms and their associated high-throughput computed analyses can represent versatile and smart systems to: (i) monitor and quantify the recruitment and interactions of the immune cells with cancer in a controlled environment, (ii) evaluate the immunogenic effects of anti-cancer therapeutic agents and (iii) evaluate the immunogenic efficacy of combinatorial regimens with respect to single agents.