How immune cells can help cancer spread, or stop it in its tracks
A free Public Lecture at Cold Spring Harbor Laboratory featuring Mikala Egeblad, Ph.D. and Sylvia Adams, M.D.
Cell Press Webinar: Cancer, Inflammation, and Immunity
In this Cell Press webinar, Douglas T. Fearon and Mikala Egeblad discuss this interplay, exploring the immunological consequences of cancer-induced cachexia as well as the effects of neutrophil extracellular traps on metastasis. Originally aired on May 17, 2017.
CSHL Assistant Professor Mikala Egeblad, Ph.D. – Breast Cancer Public Lecture
Mikala’s Public Lecture “Breast Cancer in living color: Inspiring new treatments” at Cold Spring Harbor Laboratory – March 11, 2014
CSHL Researcher Mikala Egeblad Wins $2.5 Million To Help Demystify Cancer
Mikala talks to MyLITV about the lab’s ongoing efforts to overcome therapy resistance in breast cancer. Released October 1, 2014
Structural nuclear changes during doxorubicin-induced cell death
Visualization and tracking of nuclear morphology in an MMTV-PyMT;ACTB-ECFP;H2B-EGFP mouse treated with doxorubicin. Dead cells were labeled with propidium iodide (PI). Two main cell death processes can be observed: necrotic-like cell death, where nuclear morphology is maintained when cells become PI-positive (white arrow); and apoptotic-like cell death, where nuclei undergo fragmentation before eventually becoming PI-positive (yellow arrow). The time indicated on the movie is +24 hr after doxorubicin treatment. Scale bar: 20 microm.
From Nakasone et al. (2012) Cancer Cell, 21(4):488-503.
Single cell death in a chemotherapy-treated tumor
Visualization of cell death in an MMTV-PyMT;ACTB-ECFP;c-fms-EGFP mouse. Cell death can be observed at the single cell level as the appearance of propidium iodide (PI)-positivity in previously PI-negative, ECFP-positive cancer cells (arrow). Cancer cell death is followed by infiltration of myeloid cells (green) into the dying tissue. The time indicated on the movie is +24 hr after doxorubicin treatment. Scale bar: 40 microm.
From Nakasone et al. (2012) Cancer Cell, 21(4):488-503.
Dynamics of cell death in different tumor stages in a single mouse
Cell death, tracked by propidium iodide staining (red), is observed primarily in tumors at the early carcinoma stage. Shown are three different fields of view imaged in the same MMTV-PyMT;ACTB-ECFP;c-fms-EGFP mouse. Fields correspond to a hyperplastic lesion (left), an early carcinoma (middle), and a late carcinoma (right). The time indicated on the movie is +24 hr after doxorubicin treatment. Scale bars: 100 microm.
From Nakasone et al. (2012) Cancer Cell, 21(4):488-503.
Vascular leakage in different stages in MMTV-PyMT mice
To visualize differences in vascular leakage between different tumor stages, an MMTV-PyMT;ACTB-ECFP mouse was intravenously injected with 10 kD dextran (red) and 2 MD dextran (green). Shown are three different fields of view imaged in the same mouse, corresponding to hyperplasia (left), early carcinoma (middle), and late carcinoma (right). The early carcinoma showed the greatest degree of vascular leakage, with the hyperplasia showing limited leakage and the late carcinoma showing leakage primarily at the tumor-stroma border. Time after injection with dextrans is indicated. Scale bars: 100 microm.
From Nakasone et al. (2012) Cancer Cell, 21(4):488-503.
Doxorubicin is distributed well in a medium sized tumor lesion
Doxorubicin is well distributed in a medium sized tumor lesion with uptake of the drug in the nuclei of cells in the lesion. An MMTV-PyMT mouse was injected intravenously with AngioSPARK 680 (green) to label vasculature and doxorubicin (red). Time after injection with doxorubicin is indicated. Scale bar: 100 microm.
From Nakasone et al. (2012) Cancer Cell, 21(4):488-503.
Doxorubicin is distributed poorly in a large tumor lesion
The extravascular space is poorly penetrated by doxorubicin in large tumors. An MMTV-PyMT mouse was injected intravenously with AngioSPARK 680 (green), to label the vasculature, and doxorubicin (red). Time after injection with doxorubicin is indicated. Scale bar: 100 microm.
From Nakasone et al. (2012) Cancer Cell, 21(4):488-503.
Myeloid cell infiltration to areas of doxorubicin-induced cell death
Myeloid cells (green) are shown infiltrating an area of massive cell death (red; propidium iodide staining) in an MMTV-PyMT;ACTB-ECFP;c-fms-EGFP mouse treated with doxorubicin. The time indicated on the movie is +24 hr after doxorubicin treatment. Scale bar: 100 microm.
From Nakasone et al. (2012) Cancer Cell, 21(4):488-503.
Myeloid cell infiltration and homing to necrotic debris
Myeloid cells (green) infiltrate into areas where necrotic debris (red) and dextran (blue) were injected into the mammary gland of a non-tumor-bearing c-fms-EGFP mouse (right). A field injected with only dextran was imaged in parallel in the same mouse (left). Time after injection of necrotic debris is indicated. Scale bars: 100 microm.
From Nakasone et al. (2012) Cancer Cell, 21(4):488-503.
Uptake of dead cell material after chemotherapy-induced cell death
Imaging of nuclear changes and uptake of dead cell material after chemotherapy-induced cell death. This triple transgenic MMTV-PyMT;ACTB-ECFP;H2B-EGFP animal was injected with doxorubicin prior to imaging. Nuclear morphology (green), as tracked by expression of an H2B-EGFP fusion protein allows for direct visualization of chemotherapy-induced nuclear structural changes. The animal received half-hourly i.p. injections of PI (red) for the duration of the imaging session to label dead and dying cells. Images were acquired using a high magnification objective lens (40, NA 1.1, water lens). The lack of major structural changes prior to labeling with PI and the change in the nuclear morphology and loss of GFP signal is indicative of necrosis-like cell death. The dead material is seen being taken up by a cell with a large nucleus, which is typical of macrophages. Time indicated is time after doxorubicin treatment +24 hours. Scale bar = 10 microm.
From Nakasone et al., (2013) J Vis Exp. (73). doi: 10.3791/50088.
Pre-treatment with pertussis toxin reduces myeloid cell infiltration and homing to necrotic debris
Pre-treatment with pertussis toxin, a Gi protein-coupled receptor inhibitor, impedes the recruitment of myeloid cells (green) into areas where necrotic debris (red) was injected into the mammary gland of an untreated, non-tumor-bearing c-fms-EGFP mouse. Time after injection of necrotic debris is indicated. Scale bar: 50 microm.
From Nakasone et al. (2012) Cancer Cell, 21(4):488-503.
c-fms+ myeloid cell migration in response to wounding
c-fms+ myeloid cells migrate in response to wounding on the surface of a normal mammary gland. Myeloid cells (green) are seen migrating in the mammary adipogenic stroma in a c-fms-EGFP mouse.
From Ewald et al. (2009) Chapter 23 in: Live Cell Imaging, A Laboratory Manual. 2nd edition. Cold Spring Harbor Laboratory Press. pp. 419-439.
c-fms+ myeloid cells respond to injection of E. coli in a mammary gland
c-fms+ myeloid cells respond to injection of E. coli in a mammary gland. Myeloid cells (green) are seen migrating and ingesting rhodamine-conjugated E.coli (red) in the mammary adipogenic stroma in a c-fms-EGFP mouse.
Unpublished.
c-fms+ myeloid cells migrate in the normal mammary gland
c-fms+ myeloid cells migrate in the normal mammary gland. c-fms+ cells (green) are seen migrating in the mammary adipogenic stroma and along an epithelial duct (blue) in an ACTB-ECFP;c-fms-EGFP mouse. c-fms+ cells also flow through blood vessels on top of the epithelial duct (black on the blue epithelium) and in the top of the field (recognizable as the green myeloid cells that pass by in single frames).
From Egeblad et al. (2008) Dis. Model. Mech., 1 155-167.
c-fms+ myeloid cells in the tumor microenvironment
Infiltration and migration of c-fms+ myeloid cells in the tumor microenvironment. c-fms+ myeloid cells (green) migrate at the border of a late carcinoma (bottom of field), whereas migration within the tumor mass (blue) mostly occurs along blood vessels and in stromal patches. Imaging in an MMTV-PyMT;ACTB-ECFP;c-fms-EGFP mouse. c-fms+ cells are also seen flowing through blood vessels inside the lesion (black areas on the surrounding background of blue cancer cells).
From Egeblad et al. (2008) Dis. Model. Mech., 1 155-167.
Myeloid cells extravasate and infiltrate late carcinoma lesions
Myeloid cells extravasate and infiltrate late carcinoma lesions. Two myeloid cells (green, marked with white cubes) that already have infiltrated the cancer cell masses (blue) are largely immobile while a third cell extravasates from a blood vessel in the right side of the field and infiltrates the tumor (following points A-J, from the late carcinoma shown in Movie 4, at higher magnification). The movie is shown first with and then without marking. Large tick marks are 20 microm apart.
From Ewald et al. (2009) Chapter 23 in: Live Cell Imaging, A Laboratory Manual. 2nd edition. Cold Spring Harbor Laboratory Press. pp. 419-439.
Extravasation and stromal cell uptake of fluorescent dextran in tumors
Extravasation and stromal cell uptake of fluorescent dextran in tumors. An MMTV-PyMT mouse was injected i.v. with 10kD Alexa-Fluor-647-conjugated dextran (red), 70kD rhodamine-conjugated dextran (green) and non-targeted quantum dots (blue).
From Sounni et al. (2010) Dis. Model. Mech., 3, 317-32.
Gr1+ myeloid cells are a patrolling subpopulation of the myeloid cells
Gr1+/c-fms+ myeloid cells (red/green) patrol in tumor vasculature (yellow) in the tumor-stroma border area while c-fms+/dextran+ macrophages (green/yellow) do not. Alexa-Fluor-647-conjugated anti-Gr1 antibodies (red) were coinjected, i.v., with 70 kD rhodamine-conjugated dextran (yellow) in an MMTV-PyMT;ACTB-ECFP;c-fms-EGFP mouse. Dextran first labeled the blood vessels but was taken up by macrophages over time. ECFP (blue), mostly labels epithelial cells. The movie is shown first with and then without the dextran channel. Time indicates time after injection of antibody and dextran.
From Egeblad et al. (2008) Dis. Model. Mech., 1 155-167.
CD11c+ dendritic-like cells migrate at the tumor-stroma border
CD11c+ dendritic-like cells migrate at the tumor-stroma border. CD11c+ cells (green), expressing EGFP on their membranes as a fusion protein with the diphtheria toxin receptor (DTR), are seen migrating along the epithelium (blue) at the tumor-stroma border in an MMTV-PyMT;ACTB-ECFP;CD11c-DTR-EGFP mouse.
From Egeblad et al. (2008) Dis. Model. Mech., 1 155-167.
Carcinoma-associated fibroblasts in 3 tumor regions
Carcinoma-associated fibroblasts in three different tumor regions in the same mouse, imaged before and after injection of dextran. Left panel: Fibroblast specific protein1 (FSP1)+ cells (green) are non-migratory at the border of a hyperplastic lesion (blue). Middle panel: a few migratory FSP1+ cells are seen at the border of an early carcinoma (blue). Right panel: Most FSP1+ cells that have infiltrated the carcinoma mass are non-migratory. Imaged in an MMTV-PyMT;ACTB-ECFP;Fsp1+/+.EGFP mouse injected intravenously 1 hours 41 minutes after imaging was initiated with rhodamine-conjugated dextran (red) to mark first blood vessels and then macrophages (FSP1+/dextran+; green/red).
From Egeblad et al. (2008) Dis. Model. Mech., 1 155-167.
Migration of CXCR6+ activated/memory T-cells in tumors
Migration of CXCR6+ activated/memory T-cells in the tumor microenvironment. CXCR6+ activated/memory T-cells (green) migrate in proximity to blood vessels (red) in a live MMTV-PyMT;ACTB-ECFP;CXCR6EGFP mouse injected intravenously rhodamine-conjugated dextran (red) mark blood vessels. Cancer cells are labeled blue.
From Egeblad et al. (2010) Dev. Cell, 18 884-901
Migration of CXCR6+ activated/memory T-cells & hypoxia
Migration of CXCR6+ activated/memory T-cells is stopped in response to hypoxia. T-cells (green) are seen migrating in a live MMTV-PyMT;ACTB-ECFP;CXCR6EGFP mouse. The same field is shown during normoxia (21% inhaled oxygen), hypoxia (7% inhaled oxygen) and re-established normoxia (21% oxygen). Cancer cells are labeled blue.
From Egeblad et al. (2010) Dev. Cell, 18 884-901.
Foxp3+ regulatory T cells migrate along blood vessels
Foxp3+ regulatory T cells migrate along blood vessels and at the tumor-stroma border. Regulatory T cells (green), expressing EGFP as a fusion protein to the transcription factor Foxp3 are seen migrating along a major vessel in an MMTV-PyMT;ACTB-ECFP;Foxp3EGFP mouse. Cancer cells are labeled blue.
From Egeblad et al. (2008) Dis. Model. Mech., 1 155-167.
Migration of Foxp3+ regulatory T-lymphocytes & hypoxia
Migration of Foxp3+ regulatory T-lymphocytes is stopped in response to hypoxia. Regulatory T-lymphocytes (green), expressing EGFP as a fusion protein with the transcription factor Foxp3, are seen migrating in a live MMTV-PyMT;ACTB-ECFP;Foxp3EGFP mouse. The same field is shown during normoxia (21% inhaled oxygen), hypoxia (7% inhaled oxygen) and re-established normoxia (21% oxygen). Cancer cells are labeled blue.
Unpublished.
Drug delivery into a tumor
This is a double-transgenic MMTV-PyMT;ACTB-ECFP animal in which cancer cells are labeled in blue through expression of ECFP. The animal was injected with a 2 MD FITC-conjugated dextran (green) during the imaging session to visualize how drugs reach tumors after i.v. injection. Scale bar = 100 microm.
From Nakasone et al. (2013) J Vis Exp. (73). doi: 10.3791/50088.
Drug infiltration into the tumor tissue
A triple transgenic C3(1)-Tag;ACTB-ECFP;c-fms-EGFP animal in which cancer cells are labeled in blue through expression of ECFP and myeloid cells labeled green through expression of EGFP was injected i.v. with an Alexa Fluor® 647-conjugated 10 kd dextran (red). The field of view is shown starting immediately after injection with dextran. The dextran initially labels the vasculature, rapidly extravasates into the tumor tissue, and is finally taken up by macrophages. Scale bar = 100 microm.
From Nakasone et al. (2013) J Vis Exp. (73). doi: 10.3791/50088.
Imaging of nuclear changes after chemotherapy-induced cell death
This triple transgenic MMTV-PyMT;ACTB-ECFP;H2B-EGFP animal was injected with doxorubicin prior to imaging. Nuclear morphology (green), as tracked by expression of an H2B-EGFP fusion protein allows for direct visualization of chemotherapy-induced nuclear structural changes typical of apoptosis as seen for the cell in the top right corner. The animal received half-hourly i.p. injections of PI (red) for the duration of the imaging session to label dead and dying cells. Time indicated is time after doxorubicin treatment +24 hours. Images were acquired using a high magnification objective lens (40, NA 1.1, water lens). Scale bar = 15 microm.
From Nakasone et al (2013) J Vis Exp. (73). doi: 10.3791/50088.
