Tumor Dormancy

H. Kim Lyerly, M.D., F.A.C.S.Duke University

Impact of Breast Cancer Subtypes and Treatment on Survival: An Analysis Spanning Two DecadesKaplan-Meier Curve for Breast Cancer Mortality

Haque et al. Cancer Epidemiol Biomarkers Prev . 2012 October ; 21(10): 1848–1855.

ER+PR+

ER-PR- Reactivation

of “dormant”

tumor?

Tumor growth in actual patients reflects a “dormant” state in some

C L Chaffer, R A Weinberg Science 2011;331:1559-1564

Dormant cells have colonized body

• How do breast cancer cells get into the bone?• Where in the bone do they reside during the years prior to relapse?• If the have colonized bone at the time of diagnosis, how can we prevent

micrometastases from becoming clinically significant macrometastases?

How Do Malignant Cells Get Into the Bone or Bone Marrow ?

Leukocytes As a Model for Hematogenous Metastasis

• Breast cancer cells express multiple E-selectin ligands• They express CXCR4; CXCR4 expression correlates with late

relapse (Price et al., Sci Trans Med 2016)Adapted from Immunobiology, 5th Ed., Janeway et al.

Models of dormancy control

Tumor cells evade the immune system and expand:

Not recognized Immune system

is modulated Tumor cells

resistant to immune killing

GROWTH BALANCED BY APOPTOSIS

GROWTH LIMITED BY ANGIOGENISIS

GROWTH LIMITED BY IMMUNE SYSTEM

Disseminated cells had two types of “dormancy”- “cellular” and “population”

Goss and Chambers, Nat Rev Cancer 2010

CELLULAR DORMANCY

POPULATION(of cells)

DORMANCY

DISSEMINATED METASTATIC

Parallels between stem cell regulation & regulation of dormant disseminated tumour cells by the perivascular niche.

Properties of dormant disseminated tumour cells are conferred by tissue-specific perivascular niches.

E-Selectin and SDF-1 are Expressed by the Bone Marrow Vasculature in Discrete, Sinusoidal and Peri-Sinusoidal Regions

Sipkins, et al. Nature 2005

Blood pool (LMW Dextrans)E-selectin

BCCs Enter Bone Through the BM Sinusoidal Vasculature

Patient Bone Marrow Biopsies Show Micrometastatic Disease is “Dormant” and

Located in SDF-1+ Sinusoidal Niches

Properties of dormant disseminated tumour cells are conferred by tissue-specific perivascular niches.

Resistance to: Immune recognition Immune response Immune mediated

destruction

Antigens (what is recognized by the immune system) are generated from degraded proteins and presented on cell surface to be recognized by T cells

Tumor cell or cell infected with a virus

Antigen Presenting Cell,

(A Normal Component Of The Immune

System)

Cytotoxic T Cell Helper T Cell

Both antigen and co-stimulation are required to elicit an immune response

Molecular Mechanisms of Down Modulation

Combinations of therapies may be required for immune therapy to overcome immune-suppressive in all cases

microenvironment.

#1

#2 #3

#4

Schematic representations of some immune escape mechanisms used by tumour cells.

Another form of immune escape is due to loss of antigen and rise in alternative peptide antigens.

Immune strategies

• It is widely accepted that we must now consider the immunosuppressive micro-environment of metastatic lesions to enhance immune therapies

• What of the micro-environment of the “disseminated” tumor cells (DTC)? Is it the same or different than the metastatic tumor microenvironment? The niche for dormant DTC is thought to be different….

E0771 mammary tumor cells in C57/BL6 mice model to probe DTC interactions with engineered T cells in vivo.

• A model of breast cancer that allows expression a defined antigen in tumor cells, implantation of tumor cells in immune competent mice, and analysis of antigen specific T cells recognizing, interacting, and eliminating DTCs derived from the tumor.

• OVA (a model antigen) and all associated reagents have been developed in C57/BL6 mice, so we determined that the C57/BL6-derived mammary cancer line E0771 generates detectable DTCs and could be used as a model.

The E0771 Model of dormant DTC

B. BM whole mounts generated from femurs of these mice, stained, and imaged to identify dormant (vimentin-positive, Ki67-negative) DTCs (ii, iii). Vimentin-positive cells are not present in the marrow of uninoculated, control mice (i; scale bar = 20μm). C. DTCs present in BM of inoculated mice can reconstitute tumors, as they form luciferase-positive colonies once isolated from BM and expanded in 3D culture.

A. Hundreds of GFP-luc E0771 cells detected in BM flushed from femur of C57/BL6 mice inoculated with E0771 cells in the mammary fat pad (MFP) 4-weeks prior.

OVA is detected by qPCR specifically in E0771 mammary tumor cells engineered to specifically express the antigen ovalbumin (OVA) and not in wild-type control cells.

OT1 cells eliminate OVA-expressing DTCs isolated from bone marrow of mice inoculated with E0771-OVA cells in culture. Increasing the ratio of OT1 cells:E0771 DTCs isolated from the bone marrow of E0771-OVA inoculated C57/BL6 mice results in enhanced cytotoxicity. This demonstrates the feasibility of using the OVA system to functionally probe DTC-T cell interactions.

The E0771 OVA Model of Immune Recognition

Future Objectives

• Metastatic cancer remains a clinical challenge.• Can we understand how the DTC escape

immune recognition and elimination?• Can we develop a non-toxic strategy to

eliminate DTC from residing in the body?• Can we develop a non-toxic therapy to

prevent DTC from developing into clinically relevant metastasis?

Thanks

Zach Hartman, Ph.D.Duke University

Dorothy Sipkins, M.D., Ph.D.Duke University

Cyrus M. Ghajar, Ph.D.Fred Hutchinson Cancer Research Center