Isolation of mouse mammary carcinoma-derived macrophages and cancer cells for co-culture assays

We recently established an in vitro co-culture system in which monophosphoryl lipid A + interferon-γ (MPLA+IFNγ)-treated tumor-associated macrophages (TAMs) killed cancer cells. Here, we describe a step-by-step protocol for isolating TAMs and cancer cells from mouse primary mammary carcinomas, the setup of the co-culture system, and the image acquisition approach. The technical difficulties in the co-culture assay involve isolating pure TAMs and cancer cells from the same tumor and staining them with different dyes to track the macrophages’ tumoricidal activity. For complete details on the use and execution of this protocol, please refer to Sun et al. (2021).(1

Matrix Metalloproteinase 13 Is Induced in Fibroblasts in Polyomavirus Middle T Antigen-Driven Mammary Carcinoma without Influencing Tumor Progression

Matrix metalloproteinase (MMP) 13 (collagenase 3) is an extracellular matrix remodeling enzyme that is induced in myofibroblasts during the earliest invasive stages of human breast carcinoma, suggesting that it is involved in tumor progression. During progression of mammary carcinomas in the polyoma virus middle T oncogene mouse model (MMTV-PyMT), Mmp13 mRNA was strongly upregulated concurrently with the transition to invasive and metastatic carcinomas. As in human tumors, Mmp13 mRNA was found in myofibroblasts of invasive grade II and III carcinomas, but not in benign grade I and II mammary intraepithelial neoplasias. To determine if MMP13 plays a role in tumor progression, we crossed MMTV-PyMT mice with Mmp13 deficient mice. The absence of MMP13 did not influence tumor growth, vascularization, progression to more advanced tumor stages, or metastasis to the lungs, and the absence of MMP13 was not compensated for by expression of other MMPs or tissue inhibitor of metalloproteinases. However, an increased fraction of thin collagen fibrils was identified in MMTV-PyMT;Mmp13−/− compared to MMTV-PyMT;Mmp13+/+ tumors, showing that collagen metabolism was altered in the absence of MMP13. We conclude that the expression pattern of Mmp13 mRNA in myofibroblasts of invasive carcinomas in the MMTV-PyMT breast cancer model recapitulates the expression pattern observed in human breast cancer. Our results suggest that MMP13 is a marker of carcinoma-associated myofibroblasts of invasive carcinoma, even though it does not make a major contribution to tumor progression in the MMTV-PyMT breast cancer model

A Bivalent Activatable Fluorescent Probe for Screening and Intravital Imaging of Chemotherapy‐Induced Cancer Cell Death

The detection and quantification of apoptotic cells is a key process in cancer research, particularly during the screening of anticancer therapeutics and in mechanistic studies using preclinical models. Intravital optical imaging enables high-resolution visualisation of cellular events in live organisms; however, there are few fluorescent probes that can reliably provide functional readouts in situ without interference from tissue autofluorescence. Here we report the design and optimisation of the fluorogenic probe Apotracker Red for real-time detection of cancer cell death. The strong fluorogenic behaviour, high selectivity, and excellent stability of Apotracker Red make it a reliable optical reporter for the characterisation of the effects of anticancer drugs in cells in vitro and for direct imaging of chemotherapy-induced apoptosis in vivo in mouse models of breast cancer

Cancer cell CCR2 orchestrates suppression of the adaptive immune response.

C-C chemokine receptor type 2 (CCR2) is expressed on monocytes and facilitates their recruitment to tumors. Though breast cancer cells also express CCR2, its functions in these cells are unclear. We found that Ccr2 deletion in cancer cells led to reduced tumor growth and approximately twofold longer survival in an orthotopic, isograft breast cancer mouse model. Deletion of Ccr2 in cancer cells resulted in multiple alterations associated with better immune control: increased infiltration and activation of cytotoxic T lymphocytes (CTLs) and CD103+ cross-presenting dendritic cells (DCs), as well as up-regulation of MHC class I and down-regulation of checkpoint regulator PD-L1 on the cancer cells. Pharmacological or genetic targeting of CCR2 increased cancer cell sensitivity to CTLs and enabled the cancer cells to induce DC maturation toward the CD103+ subtype. Consistently, Ccr2-/- cancer cells did not induce immune suppression in Batf3-/- mice lacking CD103+ DCs. Our results establish that CCR2 signaling in cancer cells can orchestrate suppression of the immune response

Activating a collaborative innate-adaptive immune response to control breast and ovarian cancer metastasis

Many cancers recruit monocytes/macrophages and polarize them into tumor-associated macrophages (TAMs). TAMs promote tumor growth and metastasis and inhibit cytotoxic T cells. Yet, macrophages can also kill cancer cells after polarization by e.g., lipopolysaccharide (LPS, a bacteria-derived toll-like receptor 4 [TLR4] agonist) and interferon gamma (IFNγ). They do so via nitric oxide (NO), generated by inducible NO synthase (iNOS). Altering the polarization of macrophages could therefore be a strategy for controlling cancer. Here, we show that monophosphoryl lipid A (MPLA, a derivative of LPS) with IFNγ activated macrophages isolated from metastatic pleural effusions of breast cancer patients to kill the corresponding patients’ cancer cells in vitro. Importantly, intratumoral injection of MPLA with IFNγ not only controlled local tumor growth but also reduced metastasis in mouse models of luminal and triple negative breast cancers. Furthermore, intraperitoneal administration of MPLA with IFNγ reprogrammed peritoneal macrophages, suppressed metastasis, and enhanced the response to chemotherapy in the ID8-p53−/− ovarian carcinoma mouse model. The combined MPLA+IFNγ treatment reprogrammed the immunosuppressive microenvironment to be immunostimulatory by recruiting leukocytes, stimulating type I interferon signaling, decreasing tumor-associated (CD206+) macrophages, increasing tumoricidal (iNOS+) macrophages, and activating cytotoxic T cells through macrophage-secreted interleukin 12 (IL-12) and tumor necrosis factor α (TNFα). Both macrophages and T cells were critical for the anti-metastatic effects of MPLA+IFNγ. MPLA and IFNγ are already used individually in clinical practice, so our strategy to engage the anti-tumor immune response, which requires no knowledge of unique tumor antigens, may be ready for near-future clinical testing

Cancer cell CCR2 orchestrates suppression of the adaptive immune response

ABSTRACT C-C chemokine receptor type 2 (CCR2) is expressed on monocytes and facilitates their recruitment to tumors. Although breast cancer cells also express CCR2, its functions in these cells are unclear. We found that Ccr2 deletion in cancer cells led to reduced tumor growth and ∼2-fold longer survival in an orthotopic isograft breast cancer mouse model. Deletion of Ccr2 in cancer cells resulted in multiple alterations associated with better immune control: increased infiltration and activation of cytotoxic T lymphocytes (CTLs) and CD103+ cross-presenting dendritic cells (DCs), as well as upregulation of MHC class I and downregulation of checkpoint regulator PD-L1 on the cancer cells. Pharmacological inhibition of CCR2 increased cancer cell sensitivity to CTLs and enabled cancer cells to induce DC maturation toward the CD103+ subtype. Consistently, Ccr2 -/- cancer cells did not induce immune suppression in Batf3 -/- mice lacking the CD103+ DC subtype. Our results establish that CCR2 signaling in cancer cells can orchestrate suppression of the immune response. Summary C-C chemokine receptor type 2 (CCR2) expressed on monocytes facilitates their recruitment to tumors. Here, CCR2 signaling in cancer cells is shown to suppress immune control of tumors, in part by reducing CD103+ dendritic cell recruitment

Disulfiram inhibits neutrophil extracellular trap formation protecting rodents from acute lung injury and SARS-CoV-2 infection.

Severe acute lung injury has few treatment options and a high mortality rate. Upon injury, neutrophils infiltrate the lungs and form neutrophil extracellular traps (NETs), damaging the lungs and driving an exacerbated immune response. Unfortunately, no drug preventing NET formation has completed clinical development. Here, we report that disulfiram -an FDA-approved drug for alcohol use disorder- dramatically reduced NETs, increased survival, improved blood oxygenation, and reduced lung edema in a transfusion-related acute lung injury (TRALI) mouse model. We then tested whether disulfiram could confer protection in the context of SARS-CoV-2 infection, as NETs are elevated in patients with severe COVID-19. In SARS-CoV-2-infected golden hamsters, disulfiram reduced NETs and perivascular fibrosis in the lungs, and downregulated innate immune and complement/coagulation pathways, suggesting that it could be beneficial for COVID-19 patients. In conclusion, an existing FDA-approved drug can block NET formation and improve disease course in two rodent models of lung injury for which treatment options are limited

Iraq/Afghanistan war lung injury reflects burn pits exposure

This descriptive case series retrospectively reviewed medical records from thirty-one previously healthy, war-fighting veterans who self-reported exposure to airborne hazards while serving in Iraq and Afghanistan between 2003 and the present. They all noted new-onset dyspnea, which began during deployment or as a military contractor. Twenty-one subjects underwent non-invasive pulmonary diagnostic testing, including maximum expiratory pressure (MEP) and impulse oscillometry (IOS). In addition, five soldiers received a lung biopsy; tissue results were compared to a previously published sample from a soldier in our Iraq Afghanistan War Lung Injury database and others in our database with similar exposures, including burn pits. We also reviewed civilian control samples (5) from the Stony Brook University database. Military personnel were referred to our International Center of Excellence in Deployment Health and Medical Geosciences, Donald and Barbara Zucker School of Medicine at Hofstra/Northwell under the auspices of Northwell IRB: 17-0140-FIMR Feinstein Institution for Medical Research "Clinicopathologic characteristics of Iraq Afghanistan War Lung Injury." We retrospectively examined medical records, including exposure data, radiologic imaging, and non-invasive pulmonary function testing (MGC Diagnostic Platinum Elite Plethysmograph) using the American Thoracic Society (ATS) standard interpretation based on Morgan et al., and for a limited cohort, biopsy data. Lung tissue, when available, was examined for carbonaceous particles, polycyclic aromatic hydrocarbons (Raman spectroscopy), metals, titanium connected to iron (Brookhaven National Laboratory, National Synchrotron Light Source II, Beamline 5-ID), oxidized metals, combustion temperature, inflammatory cell accumulation and fibrosis, neutrophil extracellular traps, Sirius red, Prussian Blue, as well as polarizable crystals/particulate matter/dust. Among twenty-one previously healthy, deployable soldiers with non-invasive pulmonary diagnostic tests, post-deployment, all had severely decreased MEP values, averaging 42% predicted. These same patients concurrently demonstrated abnormal airways reactance (X5Hz) and peripheral/distal airways resistance (D5-D20%) via IOS, averaging - 1369% and 23% predicted, respectively. These tests support the concept of airways hyperresponsiveness and distal airways narrowing, respectively. Among the five soldiers biopsied, all had constrictive bronchiolitis. We detected the presence of polycyclic aromatic hydrocarbons (PAH)-which are products of incomplete combustion-in the lung tissue of all five warfighters. All also had detectable titanium and iron in the lungs. Metals were all oxidized, supporting the concept of inhaling burned metals. Combustion temperature was consistent with that of burned petrol rather than higher temperatures noted with cigarettes. All were nonsmokers. Neutrophil extracellular traps were reported in two biopsies. Compared to our prior biopsies in our Middle East deployment database, these histopathologic results are similar, since all database biopsies have constrictive bronchiolitis, one has lung fibrosis with titanium bound to iron in fixed mathematical ratios of 1:7 and demonstrated polarizable crystals. These results, particularly constrictive bronchiolitis and polarizable crystals, support the prior data of King et al. (N. Engl. J. Med. 365:222-230, 2011) Soldiers in this cohort deployed to Iraq and Afghanistan since 2003, with exposure to airborne hazards, including sandstorms, burn pits, and improvised explosive devices, are at high risk for developing chronic clinical respiratory problems, including: (1) reduction in respiratory muscle strength; (2) airways hyperresponsiveness; and (3) distal airway narrowing, which may be associated with histopathologic evidence of lung damage, reflecting inhalation of burned particles from burn pits along with particulate matter/dust. Non-invasive pulmonary diagnostic tests are a predictor of burn pit-induced lung injury

Mouse Aortic Ring Assay: A New Approach of the Molecular Genetics of Angiogenesis

Angiogenesis, a key step in many physiological and pathological processes, involves proteolysis of the extracellular matrix. To study the role of two enzymatic families, serine-proteases and matrix metalloproteases in angiogenesis, we have adapted to the mouse, the aortic ring assay initially developed in the rat. The use of deficient mice allowed us to demonstrate that PAI-1 is essential for angiogenesis while the absence of an MMP, MMP-11, did not affect vessel sprouting. We report here that this model is attractive to elucidate the cellular and molecular mechanisms of angiogenesis, to identify, characterise or screen "pro- or anti-angiogenic agents that could be used for the treatment of angiogenesis-dependent diseases. Approaches include using recombinant proteins, synthetic molecules and adenovirus-mediated gene transfer

Senescence rewires microenvironment sensing to facilitate anti-tumor immunity

Cellular senescence involves a stable cell cycle arrest coupled to a secretory program that, in some instances, stimulates the immune clearance of senescent cells. Using an immune competent liver cancer model in which senescence triggers CD8 T cell-mediated tumor rejection, we show that senescence also remodels the cell surface proteome to alter how tumor cells sense environmental factors, as exemplified by Type II interferon (IFN-y). Compared to proliferating cells, senescent cells upregulate the IFN-y receptor, become hypersensitized to microenvironmental IFN-y, and more robustly induce the antigen presenting machinery--effects also recapitulated in human tumor cells undergoing therapy-induced senescence. Disruption of IFN-y sensing in senescent cells blunts their immune-mediated clearance without disabling the senescence state or its characteristic secretory program. Our results demonstrate that senescent cells have an enhanced ability to both send and receive environmental signals, and imply that each process is required for their effective immune surveillance