microfluidic-co-culture-models-for-dissecting-immune-response-in

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 ...

co-culture-in-vitro-systems-to-reproduce-the-cancer-immunity-cycle

Co-Culture In Vitro Systems to Reproduce the Cancer-Immunity Cycle

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between ...

Università Cattolica del Sacro Cuore

Epigenetic alterations, including dysregulated DNA methylation and histone modifications, govern the progression of colorectal cancer (CRC). Cancer cells exploit epigenetic regulation to control cellular pathways, including apoptotic and metastatic signals. Since aberrations in epigenome can be pharmacologically reversed by DNA methyltransferase and histone deacetylase inhibitors, epigenetics in combination with standard agents are currently envisaged as a new therapeutic frontier in cancer, expected to overcome drug resistance associated with current treatments. In this study, we challenged this idea and demonstrated that the combination of azacitidine and romidepsin with IFN-α owns a high therapeutic potential, targeting the most aggressive cellular components of CRC, such as metastatic cells and cancer stem cells (CSCs), via tight control of key survival and death pathways. Moreover, the antitumor efficacy of this novel pharmacological approach is associated with induction of signals of immunogenic cell death. Of note, a previously undisclosed key role of IFN-α in inducing both antiproliferative and pro-apoptotic effects on CSCs of CRC was also found. Overall, these findings open a new frontier on the suitability of IFN-α in association with epigenetics as a novel and promising therapeutic approach for CRC management. 

IFN-α potentiates the direct and immune-mediated antitumor effects of epigenetic drugs on both metastatic and stem cells of colorectal cancer.

If there is a great new hope in the treatment of cancer, the immune system is it. Innate and adaptive immunity either promote or attenuate tumorigenesis and so can have opposing effects on the therapeutic outcome. Originally described as potent antivirals, Type-I interferons (IFNs) were quickly recognized as central coordinators of tumor-immune system interactions. Type-I-IFNs are produced by, and act on, both tumor and immune cells being either host-protecting or tumor-promoting. Here, we discuss Type-I-IFNs in infectious and cancer diseases highlighting their dichotomous role and raising the importance to deeply understand the underlying mechanisms so to reshape the way we can exploit Type-I-IFNs therapeutically.

Type-I-interferons in infection and cancer: Unanticipated dynamics with therapeutic implications.

Tumor neantigens (TNAs) and tumor-associated antigens (TAAs) are crucial triggers of anticancer immune responses. Through major histocompatibility complex, such antigens activate T cells, which, by releasing interferon gamma (IFN-γ) and granzyme B (GRZB), act as crucial effectors against tumor onset and progression. However, in response to immune pressure, cancer cells use different strategies to favor the establishment of an immunosuppressive tumor microenvironment (TME). Elucidating the dynamics of tumor-host co-evolution provides novel opportunities for personalized cancer immunotherapies. The in sitro (in vitro+in situ) technology is an experimental approach involving the preparation of heterocellular cell suspensions from fresh tumors and their use in vitro. The in sitro experimental setup offers the possibility to (1) analyze immune-related parameters (e.g., quantification of cytokines released in the TME), (2) reveal the mechanism of action of drugs, and (3) unveil crucial cell-intrinsic and cell-extrinsic processes boosting anticancer immune responses. Nonetheless, the in sitro technology does not fully recapitulate the complexity of the tissue "in situ" nor models the patterns of infiltrating immune cell localization, and hence parallel experimentation should be scheduled. In this chapter we discuss in sitro technology to analyze and quantify IFN-γ and GRZB production by T cells either co-cultured with cancer cells in the presence of exogenous adjuvant stimuli (i.e., an antibody targeting the immune checkpoint programmed cell death protein 1, and recombinant calreticulin) and boosting with TAAs (i.e., the model SIINFEKL ovalbumin antigen). Specifically, we describe IFN-γ and GRZB quantification by flow cytometry, ELISA and ELISpot technologies.

Assessment of IFN-γ and granzyme-B production by in "sitro" technology.

Dendritic cells (DCs) are specialized antigen presenting cells (APCs) able to intake and crosspresent antigens (Ags) on major histocompatibility complex (MHC) class I and II molecules to T cells thus initiating primary and memory immune responses. DC-mediated Ag uptake and crosspresentation represent crucial steps toward cancer recognition and eventually elimination. Cytofluorometry is a standardized procedure to study phagocytosis. By fast and reproducible single cell measurements, flow cytometry allows for simultaneous biochemical and functional analyses of Ag intake. In this chapter, we discuss a two-color flow cytometric analysis of DC-mediated uptake of apoptotic bodies. We also show data on the adjuvanticity of Type-I-interferons (Type-I-IFNs) during Ag retention as we offer a guideline and a range of advice on sample preparation and acquisition.

Cytofluorometric assessment of dendritic cell-mediated uptake of cancer cell apoptotic bodies.

Cancer cell dormancy is a common feature of human tumors and represents a major clinical barrier to the long-term efficacy of anticancer therapies. Dormant cancer cells, either in primary tumors or disseminated in secondary organs, may reawaken and relapse into a more aggressive disease. The mechanisms underpinning dormancy entry and exit strongly resemble those governing cancer cell stemness and include intrinsic and contextual cues. Cellular and molecular components of the tumor microenvironment persistently interact with cancer cells. This dialog is highly dynamic, as it evolves over time and space, strongly cooperates with intrinsic cell nets, and governs cancer cell features (like quiescence and stemness) and fate (survival and outgrowth). Therefore, there is a need for deeper insight into the biology of dormant cancer (stem) cells and the mechanisms regulating the equilibrium quiescence--proliferation are vital in our pursuit of new therapeutic opportunities to prevent cancer from recurring. Here, we review and discuss microenvironmental regulations of cancer dormancy and its parallels with cancer stemness, and offer insights into the therapeutic strategies adopted to prevent a lethal recurrence, by either eradicating resident dormant cancer (stem) cells or maintaining them in a dormant state.

Tuning Cancer Fate: Tumor Microenvironment's Role in Cancer Stem Cell Quiescence and Reawakening.

Cancer stem cells (CSCs) are tumor subpopulations driving disease development, progression, relapse and therapy resistance, and their targeting ensures tumor eradication. CSCs display heterogeneous replication stress (RS), but the functionality/relevance of the RS response (RSR) centered on the ATR-CHK1 axis is debated. Here, we show that the RSR is efficient in primary CSCs from colorectal cancer (CRC-SCs), and describe unique roles for PARP1 and MRE11/RAD51. First, we demonstrated that PARP1 is upregulated in CRC-SCs resistant to several replication poisons and RSR inhibitors (RSRi). In these cells, PARP1 modulates replication fork speed resulting in low constitutive RS. Second, we showed that MRE11 and RAD51 cooperate in the genoprotection and mitosis execution of PARP1-upregulated CRC-SCs. These roles represent therapeutic vulnerabilities for CSCs. Indeed, PARP1i sensitized CRC-SCs to ATRi/CHK1i, inducing replication catastrophe, and prevented the development of resistance to CHK1i. Also, MRE11i + RAD51i selectively killed PARP1-upregulated CRC-SCs via mitotic catastrophe. These results provide the rationale for biomarker-driven clinical trials in CRC using distinct RSRi combinations.

Control of replication stress and mitosis in colorectal cancer stem cells through the interplay of PARP1, MRE11 and RAD51.

Cancer stem cells (CSCs) drive not only tumor initiation and expansion, but also therapeutic resistance and tumor relapse. Therefore, CSC eradication is required for effective cancer therapy. In preclinical models, CSCs demonstrated high capability to tolerate even extensive genotoxic stress, including replication stress, because they are endowed with a very robust DNA damage response (DDR). This favors the survival of DNA-damaged CSCs instead of their inhibition via apoptosis or senescence. The DDR represents a unique CSC vulnerability, but the abrogation of the DDR through the inhibition of the ATR-CHK1 axis is effective only against some subtypes of CSCs, and resistance often emerges. Here, we analyzed the impact of druggable DDR players in the response of patient-derived colorectal CSCs (CRC-SCs) to CHK1/2 inhibitor prexasertib, identifying RAD51 and MRE11 as sensitizing targets enhancing prexasertib efficacy. We showed that combined inhibition of RAD51 and CHK1 (via B02+prexasertib) or MRE11 and CHK1 (via mirin+prexasertib) kills CSCs by affecting multiple genoprotective processes. In more detail, these two prexasertib-based regimens promote CSC eradication through a sequential mechanism involving the induction of elevated replication stress in a context in which cell cycle checkpoints usually activated during the replication stress response are abrogated. This leads to uncontrolled proliferation and premature entry into mitosis of replication-stressed cells, followed by the induction of mitotic catastrophe. CRC-SCs subjected to RAD51+CHK1 inhibitors or MRE11+CHK1 inhibitors are eventually eliminated, and CRC-SC tumorspheres inhibited or disaggregated, via a caspase-dependent apoptosis. These results support further clinical development of these prexasertib-based regimens in colorectal cancer patients.

The Targeting of MRE11 or RAD51 Sensitizes Colorectal Cancer Stem Cells to CHK1 Inhibition.

Type I Interferons (IFNs) are key regulators of natural and therapy-induced host defense against viral infection and cancer. Several years of remarkable progress in the field of oncoimmunology have revealed the dual nature of these cytokines. Hence, Type I IFNs may trigger anti-tumoral responses, while leading immune dysfunction and disease progression. This dichotomy relies on the duration and intensity of the transduced signaling, the nature of the unleashed IFN stimulated genes, and the subset of responding cells. Here, we discuss the role of Type I IFNs in the evolving relationship between the host immune system and cancer, as we offer a view of the therapeutic strategies that exploit and require an intact Type I IFN signaling, and the role of these cytokines in inducing adaptive resistance. A deep understanding of the complex, yet highly regulated, network of Type I IFN triggered molecular pathways will help find a timely and immune"logical" way to exploit these cytokines for anticancer therapy.

The Yin and Yang of Type I IFNs in Cancer Promotion and Immune Activation.

Chronic viral infection and cancer are closely inter-related and are both characterized by profound alteration of tissue homeostasis. The actin cytoskeleton dynamics highly participate in tissue homeostasis and act as a sensor leading to an immune-mediated anti-cancer and anti-viral response. Herein we highlight the crucial role of actin cytoskeleton dynamics in participating in a viral mimicry activation with profound effect in anti-tumor immune response. This still poorly explored field understands the cytoskeleton dynamics as a platform of complex signaling pathways which may regulate Type I IFN response in cancer. This emerging network needs to be elucidated to identify more effective anti-cancer strategies and to further advance the immuno-oncology field which has revolutionized the cancer treatment. For a progress to occur in this exciting arena we have to shed light on actin cytoskeleton related pathways and immune response. Herein we summarize the major findings, considering the double sword of the immune response and in particular the role of Type I IFN pathways in resistance to anti-cancer treatment.

Actin Cytoskeleton Dynamics and Type I IFN-Mediated Immune Response: A Dangerous Liaison in Cancer?

Cancer stem cells (CSCs) are broadly considered immature, multipotent, tumorigenic cells within the tumor mass, endowed with the ability to self-renew and escape immune control. All these features contribute to place CSCs at the pinnacle of tumor aggressiveness and (immune) therapy resistance. The immune privileged status of CSCs is induced and preserved by various mechanisms that directly affect them (e.g., the downregulation of the major histocompatibility complex class I) and indirectly are induced in the host immune cells (e.g., activation of immune suppressive cells). Therefore, deeper insights into the immuno-biology of CSCs are essential in our pursuit to find new therapeutic opportunities that eradicate cancer (stem) cells. Here, we review and discuss the ability of CSCs to evade the innate and adaptive immune system, as we offer a view of the immunotherapeutic strategies adopted to potentiate and address specific subsets of (engineered) immune cells against CSCs.

The Immune Privilege of Cancer Stem Cells: A Key to Understanding Tumor Immune Escape and Therapy Failure.

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.

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

Cancer immunotherapy is the great breakthrough in cancer treatment as it displayed prolonged progression-free survival over conventional therapies, yet, to date, in only a minority of patients. In order to broad cancer immunotherapy clinical applicability some roadblocks need to be overcome, first among all the lack of preclinical models that faithfully depict the local tumor microenvironment (TME), which is known to dramatically affect disease onset, progression and response to therapy. In this review, we provide the reader with a detailed overview of current 3D models developed to mimick the complexity and the dynamics of the TME, with a focus on understanding why the TME is a major target in anticancer therapy. We highlight the advantages and translational potentials of tumor spheroids, organoids and immune Tumor-on-a-Chip models in disease modeling and therapeutic response, while outlining pending challenges and limitations. Thinking forward, we focus on the possibility to integrate the know-hows of micro-engineers, cancer immunologists, pharmaceutical researchers and bioinformaticians to meet the needs of cancer researchers and clinicians interested in using these platforms with high fidelity for patient-tailored disease modeling and drug discovery.

3D cancer models: One step closer to  human studies.

Immunogenic- and immune-therapies have become hot spots in the treatment of cancer. Although promising, these strategies are frequently associated with innate or acquired resistance, calling for combined targeting of immune inhibitory signals. Epigenetic therapy is attracting considerable attention as a combination partner for immune-based therapies due to its role in molding the state and fate of cancer and immune cells in the tumor microenvironment. Here, we describe epigenetic dysregulations in cancer, with a particular focus on those related to innate immune signaling and Type I interferons, and emphasize opportunities and current efforts to translate this knowledge into treatment regimens with improved clinical benefit.

Epigenetics behind tumor immunology: a mini review.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease. This is due to its aggressive course, late diagnosis and its intrinsic drugs resistance. The complexity of the tumor, in terms of cell components and heterogeneity, has led to the approval of few therapies with limited efficacy. The study of the early stages of carcinogenesis provides the opportunity for the identification of actionable pathways that underpin therapeutic resistance.

Talniflumate abrogates mucin immune suppressive barrier improving efficacy of gemcitabine and nab-paclitaxel treatment in pancreatic cancer.

A cardinal principle of oncoimmunology is that cancer cells can be eliminated by tumor-infiltrating cytotoxic CD8 T lymphocytes. This has been widely demonstrated during the last 20 years and also recently harnessed for therapy. However, emerging evidence indicates that even neoplasms showing striking initial responses to conventional and targeted (immuno)therapies often acquire resistance, resulting in tumor relapse, increased aggressiveness, and metastatization. Indeed, tumors are complex ecosystems whose malignant and nonmalignant cells, constituting the tumor microenvironment, constantly interact and evolve in space and time. Together with patient's own genetic factors, such environmental interplays may curtail antitumor immune responses leading to cancer immune evasion and natural/acquired (immuno)therapy resistance. In this context, cancer stem cells (CSCs) are thought to be the roots of therapy failure. Flow cytometry is a powerful technology that finds extensive applications in cancer biology. It offers several unique advantages as it allows the rapid, quantitative, and multiparametric analysis of cell populations or functions at the single-cell level. In this chapter, we discuss a two-color flow cytometric protocol to evaluate cancer cell immunogenicity by analyzing the proliferative and tumor-killing potential of ovalbumin (OVA)-specific CD8 OT-1 T cells exposed to OVA-expressing MCA205 sarcoma cells and their CSC counterparts.

ArticleTotal : 2	Year
Publication title Cited by 19	2021
Publication title	2024

Article	Year
IFN-α potentiates the direct and immune-mediated antitumor effects of epigenetic drugs on both metastatic and stem cells of colorectal cancer. Oncotarget\| PubMed ID: 27028869	2016
Type-I-interferons in infection and cancer: Unanticipated dynamics with therapeutic implications. Oncoimmunology\| PubMed ID: 28638743	2017
Assessment of IFN-γ and granzyme-B production by in "sitro" technology. Methods in enzymology\| PubMed ID: 31948559	2020
Cytofluorometric assessment of dendritic cell-mediated uptake of cancer cell apoptotic bodies. Methods in enzymology\| PubMed ID: 32000907	2020
Tuning Cancer Fate: Tumor Microenvironment's Role in Cancer Stem Cell Quiescence and Reawakening. Frontiers in immunology\| PubMed ID: 33193295	2020
Control of replication stress and mitosis in colorectal cancer stem cells through the interplay of PARP1, MRE11 and RAD51. Cell death and differentiation\| PubMed ID: 33531658	2021
The Targeting of MRE11 or RAD51 Sensitizes Colorectal Cancer Stem Cells to CHK1 Inhibition. Cancers\| PubMed ID: 33921638	2021
The Yin and Yang of Type I IFNs in Cancer Promotion and Immune Activation. Biology\| PubMed ID: 34571733	2021
Actin Cytoskeleton Dynamics and Type I IFN-Mediated Immune Response: A Dangerous Liaison in Cancer? Biology\| PubMed ID: 34571790	2021
The Immune Privilege of Cancer Stem Cells: A Key to Understanding Tumor Immune Escape and Therapy Failure. Cells\| PubMed ID: 34572009	2021
Type I IFNs promote cancer cell stemness by triggering the epigenetic regulator KDM1B. Nature immunology\| PubMed ID: 36002648	2022
3D cancer models: One step closer to human studies. Frontiers in immunology\| PubMed ID: 37114038	2023
Epigenetics behind tumor immunology: a mini review. Oncogene\| PubMed ID: 37604925	2023
Talniflumate abrogates mucin immune suppressive barrier improving efficacy of gemcitabine and nab-paclitaxel treatment in pancreatic cancer. Journal of translational medicine\| PubMed ID: 37996891	2023
In Vitro Evaluation of Cancer Cell Immunogenicity and Antigen-Specific T-Cell Cytotoxicity by Flow Cytometry. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 38070104	2024

Martina Musella

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