Imaging and Defining Emergent Behaviors of the Immune Response

The Immune System in Tumors

How do immune cells interact with a primary tumor?

Live slice imaging of T cell interactions with tumor and with tumor-associate myeloid cells. In this movie, blue T cells are seen to interact with myeloid immune cells that have been programmed to turn them ‘off’ (green, yellow and orange cells, so-called Tumor Associated Macrophages or TAMs) as well as with cells that can turn T cells ‘on’ (deep red cells, Dendritic Cells). Note that the TAMs get the priveledged position, just next to the tumor (tumor is in the black space in the center).  Details: Live tumor slice movie from PyMTchOVA x Cx3cr1-eGFP x Cd11c-mcherry with Day 4 adoptively transferred Actin-CFP OT-1 CD8+ T cells (Blue). DC1/2 populations labeled in red, TAM1 in green and, TAM2 in yellow. Movie spans a 55-minute imaging window. First sequence shows whole view (scale bar = 30um) for duration of time, then movie zooms (scale bar = 10um) to highlight T cell interactions with green TAM1 and yellow TAM2 cells, then subsequently red DC1/2 cells.

What does it look like when a tumor cell shows up at a new site, like a lung, and tries to establish residency? What does the tumor cell do? How does the immune system handle this?

The first few minutes of 'metastatic seeding.' Intravital imaging of pulmonary vasculature of mTmG mice (red) during injection with Hoechst- labeled B16-ZsGreen tumor cells (Green cells with blue nuclei). Evans Blue was co-injected at time of i.v. injection with tumor cells to label vascular flow. Z volumes were collected every 20 seconds and movie represents 2 minutes and 20 seconds of real time. White arrows indicate production and release of cytoplasts microparticles.

Some tumor cells cannot live in the shear environment of the lung: Spontaneous lysis of a melanoma tumor cell in lung vasculature. Intravital imaging of pulmonary vasculature of mTmG mice (red) 1 hr post-injection with Hoechst- labeled B16-ZsGreen cells. Evans Blue was co-injected at time of i.v. injection with tumor cells to label vascular flow. White arrows and text highlight the moment of lysis of a B16-ZsGreen melanoma cell (loss of green fluorescence signal), the nucleus remains intact during this even and is swept away by vascular flow. Z volumes were collected every 30 seconds and movie represents ~12 minutes of real time.

Over the next 8 hours, tumor cells can produce many many little ‘cytoplasts’, also called microparticles. Intravital imaging of pulmonary vasculature of an Actin-CFP mouse (blue) from 15 minutes until 8 hrs and 38minutes post-IV injection with Hoechst-labeled B16-ZsGreen tumor cells (green cells with blue nuclei). Z volumes were collected every 30 seconds for the first 1hr15 minutes and every 60 seconds thereafter. White arrows and text highlight various features of imaging throughout.

Most of those microparticles are ingested by immune myeloid cells in lung vasculature. Intravital imaging of pulmonary vasculature of a MacBlue mouse (blue cells are myeloid cells expressing CFP driven by a CSF1R-promoter reporter) 3 hrs and 15 minutes post-IV injection with B16-ZsGreen tumor cells (green). Green blebs shown are cytoplast microparticles. Z volumes were collected every 1min 25 seconds and movie represents ~41 minutes of real time. White arrow highlights the moment of ingestion of a microparticle by a migrating MacBlue+ myeloid cell.

Later, when only a few metastatic cells remain, they have recruited those microparticle-loaded immune cells to form a sort of ‘nest’ around them.  Tumor antigen bearing macrophages stably interact with 24hr surviving metastatic melanoma in lung. Intravital imaging of pulmonary vasculature of a MacBlue mouse x CD11c-mCherry where coexpression of CFP and mCherry label monocyte-derived Macrophages. Imaging was performed 24hrs post-injection of B16-ZsGreen tumor cells IV (green cells). Z volumes were collected every 2mins for and movie represents ~57 min of real time. Movie clearly shows the presence of CFP and CFP+ CD11c+ macrophages with ZsGreen+ inclusions surrounding this surviving metastasis.

Meanwhile, other myeloid immune cells have taken those microparticles on a ride, to the ‘regional draining lymph node’, an kind of immune organ that acts as the meeting-place for immune cells.  Tumor antigen loaded Dendritic cells interact with activated antigen specific T cells in the mediastinal LN. Live explant imaging of a mediastinal LN from a CD11c-mCherry reporter mouse, taken 72 hrs post-IV injection of with B16-ZsGreenOVA. GFP+ cells are OT-1 T cells specific to the ovalbumin model tumor antigen. Arrows highlight CD11c+ dendritic cells bearing tumor antigen (yellow) engaged in interaction with a scrum of OT-1 T cells. Z volumes were taken every 20 seconds and movie represents 30 minutes of real time.

Breast Cancer:

The Immune system is capable of recognizing and destroying many tumors. This is likely due to the ‘altered self’ nature of tumors; they are similar to normal cells but differ in the expression pattern of specific proteins and/or the generation of new ‘mutant’ proteins as a result of their transformation. Fragments of these tumor- proteins are called ‘tumor antigens’. Imaging of T cells that recognize tumor antigens in breast tumors of a mouse strain prone to breast cancer has identified a deceleration of incoming tumor-specific T cells in a specific region of the tumor. In a mouse model in which the tumors become red fluorescent as a result of their oncogenic transformation (PyMT Cherry OVA mice or “PyMTChOVA”) a unique population of phagocytic cells also become red fluorescent because they are the cells that ‘survey’ the tumor. These cells, located along the margin of the developing tumor, are then the site of the T cell deceleration. Altogether, this identifies these ‘Tumor Dendritic cells’ as partners for those T cells. Intriguingly, in vitro analysis of the partnership of these cells result in tolerization of the T cells. The movies below highlight some of these dynamics.

Direct 3D imaging of the key antigen-presenting cells in a breast tumor:  CD11c+ ‘Tumor Dendritic Cells’.
3D rendering of CD11c-YFP DC surrounding PyMTChOVA tumors.  YFP+ DCs (green) are detected surrounding PyMT tumors (red).  Surgically exposed breast tumor from PyMT ChOVA x CD11c-YFP mouse shown.  Movie shows one complete 360 rotation.  Note ‘yellow’ TuDC nearest the tumor, representing YFP+ TuDC that have ingested the red ‘mCherry’ protein from the tumors. From Engelhardt et al 2012.

The tumor-phagocytosing and tumor-marginating “TuDC” are a unique population of  cells that move more slowly as compared to cells further from the tumor.
Time-lapse movies of YFP+ TuDCs (green) surrounding PyMT tumors (red), then thresholded and masked DCs are displayed as either orange (proximal to tumor) or blue (distal to tumor).  Views are a Z projection of 337.92um (X) x 337.92um (Y) x 24um (Z) with .33um (X) x .33um (Y) x 4um (Z) resolution.  Time stamp indicates elapsed time in min:sec. From Engelhardt et al 2012.

Incoming Cytotoxic T cells Decelerate on the Margins of Tumors in a Spontaneous and Progressing Model of Breast Cancer.
Distribution, confined motility and clustering of tumor-specific T lymphocytes proximal to spontaneous tumors.  OT-I T-cells (green) are detected at the tumor site of PyMT tumors (red), then thresholded and masked T cells are displayed as either orange (proximal to tumor) or blue (distal to tumor).  Image is a Z projection of 337.92mm (X) x 337.92mm (Y) x 24mm (Z) with .33mm (X) x .33mm (Y) x 8mm (Z) resolution.  Time stamp indicates elapsed time in min:sec.  Image of surgically exposed PyMT ChOVA breast tumor 5 days post adoptive transfer of OT-I x Ub-GFP T cells. From Engelhardt et al 2012.

The TuDC often underly the decelerated T cells.
RFP+ T cells (red) are detected interacting with YFP+ DCs (green) at the site of a PyMT tumor. Image of surgically exposed PyMT ChOVA X CD11c-YFP breast tumor 5 days post adoptive transfer of OT-I x CD2 RFP T cells. Note the swarm of T cells (red) on top of a green DC (upper right) and ‘doublet’ of a red T cell that meets and associates with a green DC (lower left).  The tumor ‘nest’ is the dim ‘sphere’ a t the enter of this image and the image drifts upward to the right during image acquisition.  From Engelhardt et al 2012. 

 

Note: Some movies may not play when using the Firefox web browser. If this occurs, please use a different web browser or update your flash player.

Krummel Lab © 2018