Oncogene-Driven Induction of Orthotopic Cholangiocarcinoma in Mice.

Cholangiocarcinoma (CCA) is a malignancy affecting the epithelial cells that line the bile ducts. This cancer shows a poor prognosis and current treatments remain inefficient. Orthotopic CCA mouse models are useful for the development of innovative therapeutic strategies. Here, we describe an orthotopic model of intrahepatic CCA that can be easily induced in mice within 5 weeks at a high incidence. It is achieved by expressing two oncogenes, namely, (i) the intracellular domain of the Notch1 receptor (NICD) and (ii) AKT, in hepatocytes by means of the sleeping beauty transposon system. These plasmid vectors are delivered by hydrodynamic injection into the tail vein. The present chapter also describes how to perform magnetic resonance imaging (MRI) of the livers to visualize intrahepatic CCA nodules.

Flow Cytometry Assessment of Lymphocyte Populations Infiltrating Liver Tumors.

Tissue-resident and recruited immune cells are essential mediators of natural and therapy-induced immunosurveillance of liver neoplasia. This idea has been recently reinforced by the clinical approval of immune checkpoint inhibitors for the immunotherapy of hepatocellular carcinoma and cholangiocarcinoma. Such research progress relies on the in-depth characterization of the immune populations that are present in pre-neoplastic and neoplastic hepatic lesions. A convenient technology for advancing along this path is high-dimensional cytometry.In this chapter, we present a protocol to assess the subtype and differentiation state of hepatic lymphocyte populations by multicolor immunofluorescence staining and flow cytometry. We detail the steps required for viability assessment and immune cell phenotyping of single-cell suspensions of liver cells by means of surface and intracellular staining of more than a dozen markers of interest. This protocol does not require prior removal of debris and dead cells and allows to process multiple samples in parallel. The procedure includes the use of a fixative-resistant viability dye that allows cell fixation and permeabilization after cell surface staining and before intracellular staining and data acquisition on a flow cytometer. Moreover, we provide a panel of fluorochrome-labeled antibodies designed for the characterization of lymphocytic subsets that can be adapted to distinct experimental settings. Finally, we present an overview of the post-staining pipeline, including data acquisition on a flow cytometer and tools for post-acquisition analyses.

Isolation of Primary Mouse Hepatocytes and Non-Parenchymal Cells from a Liver with Precancerous Lesions.

In the early stages of liver carcinogenesis, rare hepatocytes and cholangiocytes are transformed into preneoplastic cells, which can progressively acquire a neoplastic phenotype, favored by the failure of natural antitumor immunosurveillance. The detailed study of both hepatic parenchymal (e.g., hepatocytes) and non-parenchymal cells (NPCs), such as immune cells, could help understand the cellular microenvironment surrounding these pre-cancerous and neoplastic lesions.Cultures of primary hepatocytes are of interest in various biomedical research disciplines, serving as an ex vivo model for liver physiology. Obtaining high viability and yield of primary mouse hepatocytes and other liver cell populations is technically challenging, thus limiting their use. In the first section of the current chapter, we introduce a protocol based on the two-step collagenase perfusion technique (by inferior vena cava) to isolate hepatocytes and, to a lower extent, NPCs and detailed the different considerations to take into account for a successful perfusion. The liver is washed by perfusion, hepatocytes are dissociated with collagenase, and different cell populations are separated by centrifugation. Various techniques have been described for the isolation of healthy and malignant hepatocytes; however, the viability and purity of the isolated cells is frequently not satisfactory. Here, we significantly optimized this protocol to reach improved yield and viability of the hepatocytes and concomitantly obtain preserved NPC populations of the liver.Within NPCs, tissue-resident or recruited immune cells are essential actors regulating hepatocarcinogenesis. However, simultaneous isolation of hepatic leukocytes together with other cell types generally yields low immune cell numbers hindering downstream application with these cells. In the second section of this chapter, as opposed to the first section primarily aiming to isolate hepatocytes, we present a tissue dissociation protocol adapted to efficiently recover leukocytes from non-perfused bulk (pre-)cancerous livers. This protocol has been optimized to be operator-friendly and fast compared to other liver processing methods, allowing easy simultaneous sample processing to retrieve hepatic (tumor-infiltrating) immune cells.

Mammary carcinoma: toward a realistic mouse model of incurable cancers.

As long as breast cancer (BC) stays under immunosurveillance, it can be controlled by treatments eliciting anticancer immune responses. However, once BC escapes immunosurveillance, it becomes therapeutically uncontrollable. A paper in the Journal for ImmunoTherapy of Cancer describes a new hormone receptor-positive BC cell line generating incurable tumors in C57BL/6 mice.

A Chemically Defined TLR3 Agonist with Anticancer Activity.

Toll-like receptor 3 (TLR3) agonists such as polyinosinic:polycytidylic acid (poly(I:C)) have immunostimulatory effects that can be taken advantage of to induce anticancer immune responses in preclinical models. In addition, poly(I:C) has been introduced into clinical trials to demonstrate its efficacy as an adjuvant and to enhance the immunogenicity of locally injected tumors, thus reverting resistance to PD-L1 blockade in melanoma patients. Here, we report the pharmacokinetic, pharmacodynamic, mechanistic and toxicological profile of a novel TLR3 agonist, TL-532, a chemically synthesized double-stranded RNA that is composed by blocks of poly(I:C) and poly(A:U) (polyadenylic - polyuridylic acid). In preclinical models, we show that TL-532 is bioavailable after parenteral injection, has an acceptable toxicological profile, and stimulates the production of multiple chemokines and interleukins that constitute pharmacodynamic markers of its immunostimulatory action. When given at a high dose, TL-532 monotherapy reduced the growth of bladder cancers growing on mice. In addition, in immunodeficient mice lacking formylpeptide receptor-1 (FPR1), TL-532 was able to restore the response of orthotopic subcutaneous fibrosarcoma to immunogenic chemotherapy. Altogether, these findings may encourage further development of TL-532 as an immunotherapeutic anticancer agent.

New hormone receptor-positive breast cancer mouse cell line mimicking the immune microenvironment of anti-PD-1 resistant mammary carcinoma.

BACKGROUND: Progress in breast cancer (BC) research relies on the availability of suitable cell lines that can be implanted in immunocompetent laboratory mice. The best studied mouse strain, C57BL/6, is also the only one for which multiple genetic variants are available to facilitate the exploration of the cancer-immunity dialog. Driven by the fact that no hormone receptor-positive (HR+) C57BL/6-derived mammary carcinoma cell lines are available, we decided to establish such cell lines. METHODS: BC was induced in female C57BL/6 mice using a synthetic progesterone analog (medroxyprogesterone acetate, MPA) combined with a DNA damaging agent (7,12-dimethylbenz[a]anthracene, DMBA). Cell lines were established from these tumors and selected for dual (estrogen+progesterone) receptor positivity, as well as transplantability into C57BL/6 immunocompetent females. RESULTS: One cell line, which we called B6BC, fulfilled these criteria and allowed for the establishment of invasive estrogen receptor-positive (ER+) tumors with features of epithelial to mesenchymal transition that were abundantly infiltrated by myeloid immune populations but scarcely by T lymphocytes, as determined by single-nucleus RNA sequencing and high-dimensional leukocyte profiling. Such tumors failed to respond to programmed cell death-1 (PD-1) blockade, but reduced their growth on treatment with ER antagonists, as well as with anthracycline-based chemotherapy, which was not influenced by T-cell depletion. Moreover, B6BC-derived tumors reduced their growth on CD11b blockade, indicating tumor sustainment by myeloid cells. The immune environment and treatment responses recapitulated by B6BC-derived tumors diverged from those of ER+ TS/A cell-derived tumors in BALB/C mice, and of ER- E0771 cell-derived and MPA/DMBA-induced tumors in C57BL/6 mice. CONCLUSIONS: B6BC is the first transplantable HR+ BC cell line derived from C57BL/6 mice and B6BC-derived tumors recapitulate the complex tumor microenvironment of locally advanced HR+ BC naturally resistant to PD-1 immunotherapy.

Crizotinib and ceritinib trigger immunogenic cell death via on-target effects.

Immunogenic cell death (ICD) has initially been discovered in the context of chemotherapy. High-dose crizotinib also stimulates ICD, as we described for non-small cell lung cancer lacking activating chromosomal aberrations of ALK or ROS1, the usual targets of crizotinib, indicating that crizotinib may act through off-target effects. However, we found that low-dose of ALK inhibitors, crizotinib and ceritinib, may stimulate ICD in anaplastic large cell lymphoma, in which ALK is activated due to a chromosomal translocation, suggesting on target ICD-promoting effects.

Pharmacological inhibitors of anaplastic lymphoma kinase (ALK) induce immunogenic cell death through on-target effects.

Immunogenic cell death (ICD) is clinically relevant because cytotoxicants that kill malignant cells via ICD elicit anticancer immune responses that prolong the effects of chemotherapies beyond treatment discontinuation. ICD is characterized by a series of stereotyped changes that increase the immunogenicity of dying cells: exposure of calreticulin on the cell surface, release of ATP and high mobility group box 1 protein, as well as a type I interferon response. Here, we examined the possibility that inhibition of an oncogenic kinase, anaplastic lymphoma kinase (ALK), might trigger ICD in anaplastic large cell lymphoma (ALCL) in which ALK is activated due to a chromosomal translocation. Multiple lines of evidence plead in favor of specific ICD-inducing effects of crizotinib and ceritinib in ALK-dependent ALCL: (i) they induce ICD stigmata at pharmacologically relevant, low concentrations; (ii) can be mimicked in their ICD-inducing effects by ALK knockdown; (iii) lose their effects in the context of resistance-conferring ALK mutants; (iv) ICD-inducing effects are mimicked by inhibition of the signal transduction pathways operating downstream of ALK. When ceritinib-treated murine ALK-expressing ALCL cells were inoculated into the left flank of immunocompetent syngeneic mice, they induced an immune response that slowed down the growth of live ALCL cells implanted in the right flank. Although ceritinib induced a transient shrinkage of tumors in lymphoma-bearing mice, irrespective of their immunocompetence, relapses occurred more frequently in the context of immunodeficiency, reducing the effects of ceritinib on survival by approximately 50%. Complete cure only occurred in immunocompetent mice and conferred protection to rechallenge with the same ALK-expressing lymphoma but not with another unrelated lymphoma. Moreover, immunotherapy with PD-1 blockade tended to increase cure rates. Altogether, these results support the contention that specific ALK inhibition stimulates the immune system by inducing ICD in ALK-positive ALCL.

MPA/DMBA-driven mammary carcinomas.

The polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene (DMBA, D) administered per os to wild-type female mice bearing slow-release medroxyprogesterone (MPA, M) pellets s.c. drives the formation of mammary carcinomas that recapitulate numerous immunobiological features of human luminal B breast cancer. In particular, M/D-driven mammary carcinomas established in immunocompetent C57BL/6 female mice (1) express hormone receptors, (2) emerge by evading natural immunosurveillance and hence display a scarce immune infiltrate largely polarized toward immunosuppression, (3) exhibit exquisite sensitivity to CDK4/CDK6 inhibitors, and (4) are largely resistant to immunotherapy with immune checkpoint blockers targeting PD-1. Thus, M/D-driven mammary carcinomas evolving in immunocompetent female mice stand out as a privileged preclinical platform for the study of luminal B breast cancer. Here, we provide a detailed protocol for the establishment of M/D-driven mammary carcinomas in wild-type C57BL/6 female mice. This protocol can be easily adapted to generate M/D-driven mammary carcinomas in female mice with most genetic backgrounds (including genetically-engineered mice).

Publisher Correction: Immunoprophylactic and immunotherapeutic control of hormone receptor-positive breast cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Immunoprophylactic and immunotherapeutic control of hormone receptor-positive breast cancer.

Hormone receptor (HR)+ breast cancer (BC) causes most BC-related deaths, calling for improved therapeutic approaches. Despite expectations, immune checkpoint blockers (ICBs) are poorly active in patients with HR+ BC, in part reflecting the lack of preclinical models that recapitulate disease progression in immunocompetent hosts. We demonstrate that mammary tumors driven by medroxyprogesterone acetate (M) and 7,12-dimethylbenz[a]anthracene (D) recapitulate several key features of human luminal B HR+HER2- BC, including limited immune infiltration and poor sensitivity to ICBs. M/D-driven oncogenesis is accelerated by immune defects, demonstrating that M/D-driven tumors are under immunosurveillance. Safe nutritional measures including nicotinamide (NAM) supplementation efficiently delay M/D-driven oncogenesis by reactivating immunosurveillance. NAM also mediates immunotherapeutic effects against established M/D-driven and transplantable BC, largely reflecting increased type I interferon secretion by malignant cells and direct stimulation of immune effector cells. Our findings identify NAM as a potential strategy for the prevention and treatment of HR+ BC.

Laminin-binding integrins are essential for the maintenance of functional mammary secretory epithelium in lactation.

Integrin dimers α3/ß1, α6/ß1 and α6/ß4 are the mammary epithelial cell receptors for laminins, which are major components of the mammary basement membrane. The roles of specific basement membrane components and their integrin receptors in the regulation of functional gland development have not been analyzed in detail. To investigate the functions of laminin-binding integrins, we obtained mutant mice with mammary luminal cell-specific deficiencies of the α3 and α6 integrin chains generated using the Cre-Lox approach. During pregnancy, mutant mice displayed decreased luminal progenitor activity and retarded lobulo-alveolar development. Mammary glands appeared functional at the onset of lactation in mutant mice; however, myoepithelial cell morphology was markedly altered, suggesting cellular compensation mechanisms involving cytoskeleton reorganization. Notably, lactation was not sustained in mutant females, and the glands underwent precocious involution. Inactivation of the p53 gene rescued the growth defects but did not restore lactogenesis in mutant mice. These results suggest that the p53 pathway is involved in the control of mammary cell proliferation and survival downstream of laminin-binding integrins, and underline an essential role of cell interactions with laminin for lactogenic differentiation.

Lurbinectedin synergizes with immune checkpoint blockade to generate anticancer immunity.

Systemic treatment with the active transcription inhibitor lurbinectedin aims at inducing tumor cell death in hyperproliferative neoplasms. Here we show that cell death induced by lurbinectedin reinstates and enhances systemic anticancer immune responses. Lurbinectedin treatment showed traits of immunogenic cell death, including the exposure of calreticulin, the release of ATP, the exodus of high mobility group box 1 (HMGB1) and type 1 interferon responses in vitro. Lurbinectedin treated cells induced antitumor immunity when injected into immunocompetent animals and treatment of transplanted fibrosarcomas reduced tumor growth in immunocompetent yet not in immunodeficient hosts. Anticancer effects resulting from lurbinectedin treatment were boosted in combination with PD-1 and CTLA-4 double immune checkpoint blockade (ICB), and lurbinectedin combined with double ICB exhibited strong antineoplastic effects. Cured animals exhibited long term immune memory effects that rendered them resistant to rechallenge with syngeneic tumors underlining the potency of combination therapy with lurbinectedin.

The metabolomic signature of extreme longevity: naked mole rats versus mice.

The naked mole-rat (Heterocephalus glaber) is characterized by a more than tenfold higher life expectancy compared to another rodent species of the same size, namely, the laboratory mouse (Mus musculus). We used mass spectrometric metabolomics to analyze circulating plasma metabolites in both species at different ages. Interspecies differences were much more pronounced than age-associated alterations in the metabolome. Such interspecies divergences affected multiple metabolic pathways involving amino, bile and fatty acids as well as monosaccharides and nucleotides. The most intriguing metabolites were those that had previously been linked to pro-health and antiaging effects in mice and that were significantly increased in the long-lived rodent compared to its short-lived counterpart. This pattern applies to α-tocopherol (also known as vitamin E) and polyamines (in particular cadaverine, N8-acetylspermidine and N1,N8-diacetylspermidine), all of which were more abundant in naked mole-rats than in mice. Moreover, the age-associated decline in spermidine and N1-acetylspermidine levels observed in mice did not occur, or is even reversed (in the case of N1-acetylspermidine) in naked mole-rats. In short, the present metabolomics analysis provides a series of testable hypotheses to explain the exceptional longevity of naked mole-rats.

Failure of immunosurveillance accelerates aging.

Immunosurveillance is generally conceived as a mechanism through which the immune system detects and eliminates (pre-)malignant cells, thus reducing the risk of developing cancer. A recent paper by Ovadya et al. demonstrates that knockout of the gene coding for perforin-1 causes accelerated accumulation of senescent cells in multiple mouse organs, thereby speeding up the aging process. These results suggest that immunosurveillance plays a much broader role in maintaining organismal health than it had been suspected.

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells.

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Organoids for Modeling Genetic Diseases.

In less than a decade, organoid systems have emerged as an innovative and valid in vitro method to mimic in vivo pathophysiology. Organoids are 3D structures constituted by multiple organ-specific cell types that self-organize and can function as miniature organs. Organoids have quickly become an important tool for basic and translational research with wide applications for disease modeling, drug screening, drug optimization, and personalized and regenerative medicine. In this review, we summarize the recent utilization of organoids for modeling human genetic diseases, a research area with promising biomedical applications.