Type I IFNs promote cancer cell stemness by triggering the epigenetic regulator KDM1B.

Cancer stem cells (CSCs) are a subpopulation of cancer cells endowed with high tumorigenic, chemoresistant and metastatic potential. Nongenetic mechanisms of acquired resistance are increasingly being discovered, but molecular insights into the evolutionary process of CSCs are limited. Here, we show that type I interferons (IFNs-I) function as molecular hubs of resistance during immunogenic chemotherapy, triggering the epigenetic regulator demethylase 1B (KDM1B) to promote an adaptive, yet reversible, transcriptional rewiring of cancer cells towards stemness and immune escape. Accordingly, KDM1B inhibition prevents the appearance of IFN-I-induced CSCs, both in vitro and in vivo. Notably, IFN-I-induced CSCs are heterogeneous in terms of multidrug resistance, plasticity, invasiveness and immunogenicity. Moreover, in breast cancer (BC) patients receiving anthracycline-based chemotherapy, KDM1B positively correlated with CSC signatures. Our study identifies an IFN-I → KDM1B axis as a potent engine of cancer cell reprogramming, supporting KDM1B targeting as an attractive adjunctive to immunogenic drugs to prevent CSC expansion and increase the long-term benefit of therapy.

Role of CT texture analysis for predicting peritoneal metastases in patients with gastric cancer.

PURPOSE: Aim of the study was to perform CT texture analysis in patients with gastric cancer (GC) to investigate potential role of radiomics for predicting the occurrence of peritoneal metastases (PM). MATERIALS AND METHODS: In this single-centre retrospective study, patients with gastric adenocarcinoma and surgically confirmed presence or absence of PM were, respectively, enrolled in group PM and group non-PM. Patients with T1-staging, previous treatment or presence of imaging artifacts were excluded from the study. Pre-operative CT examinations were evaluated. Acquisition protocol consisted of gastric distension with water, pre-contrast and arterial phases on upper abdomen and portal phase on thorax and whole abdomen. Texture analysis was performed on portal phase images: the region of interest was manually drawn along the margins of the primitive lesion on each slice and the volume of interest of the whole tumour was obtained. A total of 38 texture parameters were extracted and analysed. ROC curves were performed on significant texture features (p < 0.05). Multiple logistic regression was conducted on features with the best AUC to identify differentiating variables for both groups. RESULTS: A total of 90 patients were evaluated (group PM, n = 45; group non-PM, n = 45). T2/T3 tumours were prevalent in group non-PM, T4 was significantly associated with group PM. Significant differences between the two groups were observed for 22/38 texture parameters. Volume and GLRLM_LRHGE showed the greatest AUC in ROC curve analysis (0.737 and 0.734, respectively) and were found to be independent differentiating variables of group PM in the multiple regression analysis (OR 8.44, [95% CI, 1.52-46.8] and OR 18.99 [95% CI, 84-195.31], respectively). CONCLUSIONS: Our preliminary results suggest the potential value of CT texture analysis for predicting the risk of PM from GC, which may be helpful to stratify patients and address them to the most appropriate treatment.

Eosinophils in the Tumor Microenvironment.

Eosinophils are rare blood-circulating and tissue-infiltrating immune cells studied for decades in the context of allergic diseases and parasitic infections. Eosinophils can secrete a wide array of soluble mediators and effector molecules, with potential immunoregulatory activities in the tumor microenvironment (TME). These findings imply that these cells may play a role in cancer immunity. Despite these cells were known to infiltrate tumors since many years ago, their role in TME is gaining attention only recently. In this chapter, we will review the main biological functions of eosinophils that can be relevant within the TME. We will discuss how these cells may undergo phenotypic changes acquiring pro- or antitumoricidal properties according to the surrounding stimuli. Moreover, we will analyze canonical (i.e., degranulation) and unconventional mechanisms (i.e., DNA traps, exosome secretion) employed by eosinophils in inflammatory contexts, which can be relevant for tumor immune responses. Finally, we will review the available preclinical models that could be employed for the study of the role in vivo of eosinophils in cancer.

Basophils in Tumor Microenvironment and Surroundings.

Basophils represent approximately 1% of human peripheral blood leukocytes. Their effector functions were initially appreciated in the 1970s when basophils were shown to express the high-affinity receptor (FcεRI) for IgE and to release proinflammatory mediators (histamine and cysteinyl leukotriene C4) and immunoregulatory cytokines (i.e., IL-4 and IL-13). Basophils in the mouse were subsequently identified and immunologically characterized. There are many similarities but also several differences between human and mouse basophils. Basophil-deficient mice have enabled to examine the in vivo roles of basophils in several immune disorders and, more recently, in tumor immunity. Activated human basophils release several proangiogenic molecules such as vascular endothelial growth factor-A (VEGF-A), vascular endothelial growth factor-B (VEGF-B), CXCL8, angiopoietin 1 (ANGPT1), and hepatocyte growth factor (HGF). On the other side, basophils can exert anti-tumorigenic effects by releasing granzyme B, TNF-α, and histamine. Circulating basophils have been associated with certain human hematologic (i.e., chronic myeloid leukemia) and solid tumors. Basophils have been found in tumor microenvironment (TME) of human lung adenocarcinoma and pancreatic cancer. Basophils played a role in melanoma rejection in basophil-deficient mouse model. By contrast, basophils appear to play a pro-tumorigenic role in experimental and human pancreatic cancer. In conclusion, the roles of basophils in experimental and human cancers have been little investigated and remain largely unknown. The elucidation of the roles of basophils in tumor immunity will demand studies on increasing complexity beyond those assessing basophil density and their microlocalization in TME. There are several fundamental questions to be addressed in experimental models and clinical studies before we understand whether basophils are an ally, adversary, or even innocent bystanders in cancers.

Quality of life in non-early rectal cancer treated by neoadjuvant radio-chemotherapy and endoluminal loco-regional resection (ELRR) by transanal endoscopic microsurgery (TEM) versus laparoscopic total mesorectal excision.

BACKGROUND: In selected patients with N0 rectal cancer, endoluminal loco-regional resection (ELRR) by transanal endoscopic microsurgery (TEM) may be an alternative treatment option to laparoscopic total mesorectal excision (LTME). Aim of this study is to evaluate the short- and medium-term quality of life (QoL) from a retrospective analysis of prospectively collected data in patients with iT2-iT3 N0-N+ rectal cancer, who underwent ELRR by TEM or LTME after neoadjuvant radio-chemotherapy (n-RCT). METHODS: Thirty patients with iT2-iT3 rectal cancer who underwent ELRR by TEM (n = 15) or LTME (n = 15) were enrolled in this study. The choice for one operation or the other was made on the basis of predefined criteria. QoL was evaluated by EORTC QLQ-C30 and QLQ-CR38 questionnaires at admission, after n-RCT and 1, 6, and 12 months after surgery. RESULTS: No statistically significant differences in QoL evaluation were observed between the two groups, both at admission and after n-RCT. At 1 month after surgery, significantly better results in the ELRR group were observed by QLQ-C30 in: Nausea/Vomiting (p = 0.05), Appetite Loss (p = 0.003), Constipation (p = 0.05), and by QLQ-CR38 in: Body Image (p = 0.05), Sexual Functioning (p = 0.03), Future Perspective (p = 0.05) and Weight Loss (p = 0.036). At 6 months after surgery, a statistically significant worse impact after LTME was observed by QLQ-C30 in: Global Health Status (p = 0.05), Emotional Functioning (p = 0.021), Dyspnea (p = 0.008), Insomnia (p = 0.012), Appetite Loss (p = 0.014) and by QLQ-CR38 in Body Image (p = 0.05) and Defecation Problems (p = 0.001). At 1 year, the two groups were homogenous as assessed by QLQ-C30, whereas the QLQ-CR38 still showed better results of ELRR versus LTME in Body Image (p = 0.006), Defecation Problems (p = 0.01), and Weight Loss (p = 0.005). CONCLUSIONS: Based on the present series, in selected patients, earlier restoration of patients' functions is observed after ELRR by TEM than after LTME.

Mycobacterium tuberculosis PstS1 amplifies IFN-γ and induces IL-17/IL-22 responses by unrelated memory CD4+ T cells via dendritic cell activation.

The immunological mechanisms that modulate protection during Mycobacterium tuberculosis (Mtb) infection or vaccination are not fully understood. Secretion of IFN-γ and, to a lesser extent, of IL-17 by CD4(+) T cells plays a major role both in protection and immunopathology. Few Mtb Ags interacting with DCs affect priming, activation, and regulation of Ag-unrelated CD4(+) T-cell responses. Here we demonstrate that PstS1, a 38 kDa-lipoprotein of Mtb, promotes Ag-independent activation of memory T lymphocytes specific for Ag85B or Ag85A, two immunodominant protective Ags of Mtb. PstS1 expands CD4(+) and CD8(+) memory T cells, amplifies secretion of IFN-γ and IL-22 and induces IL-17 production by effector memory cells in an Ag-unrelated manner in vitro and in vivo. These effects were mediated through the stimulation of DCs, particularly of the CD8α(-) subtype, which respond to PstS1 by undergoing phenotypic maturation and by secreting IL-6, IL-1ß and, to a lower extent, IL-23. IL-6 secretion by PstS1-stimulated DCs was required for IFN-γ, and to a lesser extent for IL-22 responses by Ag85B-specific memory T cells. These results may open new perspectives for immunotherapeutic strategies to control Th1/Th17 immune responses in Mtb infections and in vaccinations against tuberculosis.

Novel allergic asthma model demonstrates ST2-dependent dendritic cell targeting by cypress pollen.

BACKGROUND: Cypress pollen causes respiratory syndromes with different grades of severity, including asthma. IL-33, its receptor ST2, and dendritic cells (DCs) have been implicated in human respiratory allergy. OBJECTIVE: We sought to define a new mouse model of allergy to cypress pollen that recapitulates clinical parameters in allergic patients and to evaluate the implications of DCs and the IL-33/ST2 pathway in this pathology. METHODS: BALB/c mice, either wild-type or ST2 deficient (ST2(-/-)), were sensitized and challenged with the Cupressus arizonica major allergen nCup a 1. Local and systemic allergic responses were evaluated. Pulmonary cells were characterized by means of flow cytometry. DCs were stimulated with nCup a 1 and tested for their biological response to IL-33 in coculture assays. RESULTS: nCup a 1 causes a respiratory syndrome closely resembling human pollinosis in BALB/c mice. nCup a 1-treated mice exhibit the hallmarks of allergic pathology associated with pulmonary infiltration of eosinophils, T cells, and DCs and a dominant TH2-type immune response. IL-33 levels were increased in lungs and sera of nCup a 1-treated mice and in subjects with cypress allergy. The allergen-specific reaction was markedly reduced in ST2(-/-) mice, which showed fewer infiltrating eosinophils, T cells, and DCs in the lungs. Finally, stimulation of DCs with nCup a 1 resulted in ST2 upregulation that endowed DCs with increased ability to respond to IL-33-mediated differentiation of IL-5- and IL-13-producing CD4 T cells. CONCLUSIONS: Our findings define a novel preclinical model of allergy to cypress pollen and provide the first evidence of a functionally relevant linkage between pollen allergens and TH2-polarizing activity by DCs through IL-33/ST2.

IL-33 stimulates the anticancer activities of eosinophils through extracellular vesicle-driven reprogramming of tumor cells.

Immune cell-derived extracellular vesicles (EV) affect tumor progression and hold promise for therapeutic applications. Eosinophils are major effectors in Th2-related pathologies recently implied in cancer. Here, we evaluated the anti-tumor activities of eosinophil-derived EV following activation with the alarmin IL-33. We demonstrate that IL-33-activated mouse and human eosinophils produce higher quantities of EV with respect to eosinophils stimulated with IL-5. Following incorporation of EV from IL-33-activated eosinophils (Eo33-EV), but not EV from IL-5-treated eosinophils (Eo5-EV), mouse and human tumor cells increased the expression of cyclin-dependent kinase inhibitor (CDKI)-related genes resulting in cell cycle arrest in G0/G1, reduced proliferation and inhibited tumor spheroid formation. Moreover, tumor cells incorporating Eo33-EV acquired an epithelial-like phenotype characterized by E-Cadherin up-regulation, N-Cadherin downregulation, reduced cell elongation and migratory extent in vitro, and impaired capacity to metastasize to lungs when injected in syngeneic mice. RNA sequencing revealed distinct mRNA signatures in Eo33-EV and Eo5-EV with increased presence of tumor suppressor genes and enrichment in pathways related to epithelial phenotypes and negative regulation of cellular processes in Eo33-EV compared to Eo5-EV. Our studies underscore novel IL-33-stimulated anticancer activities of eosinophils through EV-mediated reprogramming of tumor cells opening perspectives on the use of eosinophil-derived EV in cancer therapy.

Germinal Center Dark Zone harbors ATR-dependent determinants of T-cell exclusion that are also identified in aggressive lymphoma.

The germinal center (GC) dark zone (DZ) and light zone (LZ) regions spatially separate expansion and diversification from selection of antigen-specific B-cells to ensure antibody affinity maturation and B cell memory. The DZ and LZ differ significantly in their immune composition despite the lack of a physical barrier, yet the determinants of this polarization are poorly understood. This study provides novel insights into signals controlling asymmetric T-cell distribution between DZ and LZ regions. We identify spatially-resolved DNA damage response and chromatin compaction molecular features that underlie DZ T-cell exclusion. The DZ spatial transcriptional signature linked to T-cell immune evasion clustered aggressive Diffuse Large B-cell Lymphomas (DLBCL) for differential T cell infiltration. We reveal the dependence of the DZ transcriptional core signature on the ATR kinase and dissect its role in restraining inflammatory responses contributing to establishing an immune-repulsive imprint in DLBCL. These insights may guide ATR-focused treatment strategies bolstering immunotherapy in tumors marked by DZ transcriptional and chromatin-associated features.

Type I IFNs control antigen retention and survival of CD8α(+) dendritic cells after uptake of tumor apoptotic cells leading to cross-priming.

Cross-presentation is a crucial mechanism for generating CD8 T cell responses against exogenous Ags, such as dead cell-derived Ag, and is mainly fulfilled by CD8α(+) dendritic cells (DC). Apoptotic cell death occurring in steady-state conditions is largely tolerogenic, thus hampering the onset of effector CD8 T cell responses. Type I IFNs (IFN-I) have been shown to promote cross-priming of CD8 T cells against soluble or viral Ags, partly through stimulation of DC. By using UV-irradiated OVA-expressing mouse EG7 thymoma cells, we show that IFN-I promote intracellular Ag persistence in CD8α(+) DC that have engulfed apoptotic EG7 cells, regulating intracellular pH, thus enhancing cross-presentation of apoptotic EG7-derived OVA Ag by CD8α(+) DC. Notably, IFN-I also sustain the survival of Ag-bearing CD8α(+) DC by selective upmodulation of antiapoptotic genes and stimulate the activation of cross-presenting DC. The ensemble of these effects results in the induction of CD8 T cell effector response in vitro and in vivo. Overall, our data indicate that IFN-I cross-prime CD8 T cells against apoptotic cell-derived Ag both by licensing DC and by enhancing cross-presentation.

Interdisciplinary research in cancer and immunity employing biophysical approaches.

Three leading scientists Fabrizio Mattei, Kandice Tanner, and Mohit Kumar Jolly working in different continents and in different areas of cancer and immunology came together for an iScience Special Issue focused on the biophysical aspect of the tumor-immune dynamics. In this backstory, the iScience editor discusses with Mattei and Jolly their thoughts about this topic, the current state of the field, the collection of articles in this Special Issue, and the future of the research in this area in the coming years, and personal advice to aspiring young minds.

Cancer Immunology: From Molecular Mechanisms to Therapeutic Opportunities.

The gradual and more profound dissection of the molecular basis of cancer progression, carcinogenesis, and metastatic spread of cancer cells has led to more focused, effective, and targeted therapeutic approaches in the disparate types of solid and hematological tumors, particularly those with high ability to metastasize distant organs [...].

Traffic-related NO2 affects expression of Cupressus sempervirens L. pollen allergens.

INTRODUCTION AND OBJECTIVE: Traffic pollution has been recognized as directly worsening respiratory symptoms of allergic subjects, although whether urban air pollutants can also directly increase the allergenic potential of pollen has not yet been definitely proven. Therefore, the hypothesis that intra-urban air NO2 variation influences allergens expression in Cupressus sempervirens (Cs) L. pollen was tested. MATERIAL AND METHODS: Mature microsporophylls were cut from Cs trees of similar age and height (14-17 m) present in three different sites of Florence (Italy) and processed in the laboratory. Cs pollen allergens amount was determined by a semi-quantitative analysis of electrophoretically separated pollen extracts fractions. NO2 air concentrations were recorded by air monitoring stations located at a distance not exceeding 50 m from each pollen collection site, and the relative annual mean values were acquired by a publicly available database (Tuscan Regional Agency for Environment Protection). RESULTS: Expression of three major Cs pollen allergens was non-linearly correlated with mean annual NO2 concentrations. Expression peak of all major allergens considered was reached at NO2 air concentration (67µg/m3), far below the value at risk for direct effect on the respiratory health (European Union Directive 2008/50/EC). CONCLUSIONS: The findings suggest that intra-urban NO2 variations do affect the expression of Cs pollen major allergens, and an apparent low risk NO2 concentration should be regarded as indirectly harmful for increasing the allergenic potential of pollen.

Basophils from allergy to cancer.

Human basophils, first identified over 140 years ago, account for just 0.5-1% of circulating leukocytes. While this scarcity long hampered basophil studies, innovations during the past 30 years, beginning with their isolation and more recently in the development of mouse models, have markedly advanced our understanding of these cells. Although dissimilarities between human and mouse basophils persist, the overall findings highlight the growing importance of these cells in health and disease. Indeed, studies continue to support basophils as key participants in IgE-mediated reactions, where they infiltrate inflammatory lesions, release pro-inflammatory mediators (histamine, leukotriene C4: LTC4) and regulatory cytokines (IL-4, IL-13) central to the pathogenesis of allergic diseases. Studies now report basophils infiltrating various human cancers where they play diverse roles, either promoting or hampering tumorigenesis. Likewise, this activity bears remarkable similarity to the mounting evidence that basophils facilitate wound healing. In fact, both activities appear linked to the capacity of basophils to secrete IL-4/IL-13, with these cytokines polarizing macrophages toward the M2 phenotype. Basophils also secrete several angiogenic factors (vascular endothelial growth factor: VEGF-A, amphiregulin) consistent with these activities. In this review, we feature these newfound properties with the goal of unraveling the increasing importance of basophils in these diverse pathobiological processes.

Trogocytosis in innate immunity to cancer is an intimate relationship with unexpected outcomes.

Trogocytosis is a cellular process whereby a cell acquires a membrane fragment from a donor cell in a contact-dependent manner allowing for the transfer of surface proteins with functional integrity. It is involved in various biological processes, including cell-cell communication, immune regulation, and response to pathogens and cancer cells, with poorly defined molecular mechanisms. With the exception of eosinophils, trogocytosis has been reported in most immune cells and plays diverse roles in the modulation of anti-tumor immune responses. Here, we report that eosinophils acquire membrane fragments from tumor cells early after contact through the CD11b/CD18 integrin complex. We discuss the impact of trogocytosis in innate immune cells on cancer progression in the context of the evidence that eosinophils can engage in trogocytosis with tumor cells. We also discuss shared and cell-specific mechanisms underlying this process based on in silico modeling and provide a hypothetical molecular model for the stabilization of the immunological synapse operating in granulocytes and possibly other innate immune cells that enables trogocytosis.

Differential Effects of Alarmins on Human and Mouse Basophils.

Epithelial-derived alarmins (IL-33, TSLP, and IL-25) play an upstream role in the pathogenesis of asthma. Basophil-derived cytokines are a pivotal component of allergic inflammation. We evaluated the in vitro effects of IL-33, TSLP, and IL-25, alone and in combination with IL-3 on purified peripheral blood human basophils (hBaso) and bone marrow-derived mouse basophils (mBaso) in modulating the production of IL-4, IL-13, CXCL8 or the mouse CXCL8 equivalents CXCL1 and CXCL2. IL-3 and IL-33, but not TSLP and IL-25, concentration-dependently induced IL-4, IL-13, and CXCL8 release from hBaso. IL-3 synergistically potentiated the release of cytokines induced by IL-33 from hBaso. In mBaso, IL-3 and IL-33 rapidly induced IL-4 and IL-13 mRNA expression and protein release. IL-33, but not IL-3, induced CXCL2 and CXCL1 from mBaso. Differently from hBaso, TSLP induced IL-4, IL-13, CXCL1 and CXCL2 mRNA expression and protein release from mBaso. IL-25 had no effect on IL-4, IL-13, and CXCL1/CXCL2 mRNA expression and protein release even in the presence of IL-3. No synergism was observed between IL-3 and either IL-25 or TSLP. IL-3 inhibited both TSLP- and IL-33-induced CXCL1 and CXCL2 release from mBaso. Our results highlight some similarities and marked differences between the effects of IL-3 and alarmins on the release of cytokines from human and mouse basophils.

A Clonogenic Assay to Quantify Melanoma Micrometastases in Pulmonary Tissue.

Metastatic melanoma is one of the most aggressive types of cancers, diffused worldwide and with a significant percentage of lethality. The employment of animal models to test therapeutic strategies against melanoma growth and metastatic spread is of key relevance for cancer biologists. In this regard, the count of metastatic foci in murine lung tissue is one of the recognized methods to monitor macrometastases of melanoma. Here, we illustrate a clonogenic assay method to detect with high sensitivity the presence of single melanoma cells (micrometastases) at the pulmonary level when metastatic foci are still not detectable in the tissue. This method allows for high precision detection and quantification of melanoma metastatic spread to the lung at early stages.

Acidic and Hypoxic Microenvironment in Melanoma: Impact of Tumour Exosomes on Disease Progression.

The mechanisms of melanoma progression have been extensively studied in the last decade, and despite the diagnostic and therapeutic advancements pursued, malignant melanoma still accounts for 60% of skin cancer deaths. Therefore, research efforts are required to better define the intercellular molecular steps underlying the melanoma development. In an attempt to represent the complexity of the tumour microenvironment (TME), here we analysed the studies on melanoma in acidic and hypoxic microenvironments and the interactions with stromal and immune cells. Within TME, acidity and hypoxia force melanoma cells to adapt and to evolve into a malignant phenotype, through the cooperation of the tumour-surrounding stromal cells and the escape from the immune surveillance. The role of tumour exosomes in the intercellular crosstalk has been generally addressed, but less studied in acidic and hypoxic conditions. Thus, this review aims to summarize the role of acidic and hypoxic microenvironment in melanoma biology, as well as the role played by melanoma-derived exosomes (Mexo) under these conditions. We also present a perspective on the characteristics of acidic and hypoxic exosomes to disclose molecules, to be further considered as promising biomarkers for an early detection of the disease. An update on the use of exosomes in melanoma diagnosis, prognosis and response to treatment will be also provided and discussed.

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.

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments.

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle ''co-cultures/microscopy/advanced data analysis" sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.