Osteoblasts remotely supply lung tumors with cancer-promoting SiglecFhigh neutrophils. Lung tumors disrupt bone homeostasis and increase osteoblast activity and bone formation. Osteoblasts amplify tumor-associated SiglecFhigh neutrophils that promote tumor growth through angiogenesis, immunosuppression and other mechanisms. Serum from tumor-bearing mice increases osteoblast activity through elevated sRAGE, which stimulates neutrophil maturation. SiglecFhigh neutrophils correlate with poor survival in lung cancer patients. Therefore, lung tumors communicate with bone through factors like sRAGE to modulate osteoblasts and promote neutrophil-driven tumor progression.

1. Center for Systems Biology, Massachusetts General Hospital Research Institute and Harvard Medical School, Boston, MA 02114, USA; Graduate Program
in Immunology, Harvard Medical School, Boston, MA 02115, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA;
Institute of Biotechnology, Vilnius University, Vilnius, LT 10257, Lithuania; Endocrine Unit, Massachusetts General Hospital, Harvard Medical School,
Boston, MA 02114, USA; Department of Radiology, Massachusetts General Hospital, MA 02114, USA; Howard Hughes Medical Institute, Koch Institute for
Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Center for Regenerative Medicine, Massachusetts
General Hospital, Boston, MA 02114, USA; Swiss Institute for Experimental Cancer Research, School of Life Sciences, École Polytechnique Fédérale de
Lausanne, 1015 Lausanne, Switzerland; Division of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA
02115, USA.
Coach Professor: Dr. Chung-Yi Wu
Course Coordinator: Dr. Charles P. Lai
Sit-in Professor: Dr. Chi-Yu Fu
Dated: 20181017
Presenter: Gul Muneer
Science Dec 2017, 358(6367): eaal5081
DOI: 10.1126/science.aal5081
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Osteoblasts Remotely Supply Lung Tumors With
Cancer-promoting SiglecFhigh Neutrophils

2. Osteoblasts Remotely Supply Lung Tumors With
Cancer-promoting SiglecFhigh Neutrophils
Camilla Engblom, Christina Pfirschke, Rapolas Zilionis, Janaina Da Silva Martins, Stijn A. Bos, Gabriel
Courties, Steffen Rickelt, Nicolas Severe, Ninib Baryawno, Julien Faget, Virginia Savova, David Zemmour,
Jaclyn Kline, Marie Siwicki, Christopher Garris, Ferdinando Pucci, Hsin-Wei Liao, Yi-Jang Lin, Andita
Newton, Omar K. Yaghi, Yoshiko Iwamoto, Benoit Tricot, Gregory R. Wojtkiewicz, Matthias Nahrendorf,
Virna Cortez-Retamozo, Etienne Meylan, Richard O. Hynes, Marie Demay, Allon Klein, Miriam A. Bredella,
David T. Scadden, Ralph Weissleder, Mikael J. Pittet
2

3. Bone
SN Osteoblast
Osteocyte
Osteoclast
Osteoblast
Bone
Remodeling
Bone Formation Bone ResorptionBone Homeostasis
Osteoblasts Osteoclasts
Osteocalcin (Ocn)
Bone Dynamics (Formation & Resorption)
3
 Ocn is a marker for active bone formation.
 Bone is a dynamic tissue.
 Bone is constantly remodeled (formation & breakdown).
G-CSF, TPO
SDF-1, ANG-1
G-CSF: Granulocyte-colony stimulating factor
TPO: Thrombopoietin
ANG-1: Angiopoietin-1
SDF-1: Stromal Cell Derived Factor-1

4. 4
Bone
Osteoblast Niche
Progenitor cells
Bone Marrow
Vascular Niche
(HSC Inactive State & Self renewal)
Ang-1/Tie2, TPO/Mpl
Bone and Hematopoietic Niche: A Tale of Stem Cell
Bone-resident cells “also” regulate hematopoiesis & Immune cell fate.
Niche: a physiological microenvironment in which Hematopoietic stem cells (HSC) reside
Proliferation
Differentiation
G-CSF/R G-CSF/R
H-Stem Cell
Homing
Migration
SDF-1/CXCR4
Lymphocyte
Granulocyte
Erythrocyte

5. 5
Myeloid Cells in Tumor Stroma & Tumor Progression
SiglecFlow Neutrophils
Sialic acid-binding
Immunoglobulin-type lectins
 Cell surface proteins
 Binds Sialic acid
 Present on Immune cells
 Involved in cell adhesion &
cell signaling

6. ?
?
?
?
?
?
?
Tumor-associated
Hematopoietic Response?
Tumor-associated
Immune Response?
Circulating
Precursors?
Remote effect of cancer?
Tumor-infiltrating cells?
Tumor-induced bone alterations?
Remodeling of tumor shape?
? ?
Aims and Motivations of the Study
6

7. Lung Tumors Modulate Bone Density
Bisphosphonate
(OsteoSense)
On-going
Bone formation
Fluorescence Molecular Tomography
After 4 hrs
Before 24 hrs
7
KP or LLC Tumor
bearing (or Tumor-
free) Mice
OsteoSense
i.v.
Lung-tumor disrupt bone stromal activity in the absence of local metastasis.
±Adenovirus-Cre
12-14 week
KrasLSL-G12D/WT;
p53flox/flox (KP) Mice

8. Lung Tumors Increases Bone Density
Rotation
Source
Detector
µ-Computed Tomography µ-CT of Bone microarchitecture
Lung-tumor disrupt bone homeostatic activity & induce changes in bone.
8
3D reconstruction of µ-CT
Scans of spongy bone volume
BV/TV= Bone Vol./Total Vol.
CT of 10th Thoracic Vertebra of 53-year old
woman.
HU= Hounsfield Units

9. Lung Tumors Increase Osteoblasts Number
Cuboidal Shape Osteoblast
Goldner’s Trichrome Staining of femur (thigh bone)
Osteoid (gelatinous matrix) Characteristics of elevated
osteoblast activity
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Histological analysis identified more osteoblasts in KP tumor-bearing mice
Green - Mineralized Bone
Blue - Nuclei
Pink - Osteoid

10. Von Kossa Staining of femur (Bone mineralization)
Dynamic bone formation (histomorphometry)
Bone Histomorphometriy: Kinetic Data on Bone Turnover
Tumor bearing mice showed elevated mineralization and bone formation.
± Tumor-bearing mice
Calcein (i.v.)
7days
Demeclocycline (i.v.)
2days
Imaging
Analyzing System
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Black - Calcium deposits
Pink - Nuclei

11. STOP DTR
iDTR
Ocn⁺ Cells
Specific Cre
Ocn⁺ Cells
Specific Cre;Dtr
Dtr⁺ cells
DTR
Cre
Dtr⁺ cells death
DT Injection
DTR
DT
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Ocn+ Osteoblasts Regulate Lung Cancer Growth
Ocn+ cells foster lung tumor growth.
Dtr⁺ Ocn⁺ cells

12. 12
Ocn+ Osteoblasts Supply Tumor-infiltrating Neutrophils
Ocn+ cells are required for Amplification of Tumor-associated neutrophils

13. OcnCre;Dtr KP tumor-bearing Mice
OcnCre;Dtr
Parabiont
Control
Parabiont
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Tumor-infiltrating Cells Display Tumor-promoting Functions
Ocn+ cells contribute tumor-infiltrating neutrophils.
Tumor-infiltrating neutrophils display tumor promoting functions.

14. 14
Ocn+ Cell-driven Neutrophils
Ocn+ cells-driven neutrophils show discrete phenotypes & localize proximal to tumor cells
Neutrophils are heterogenous
- Molecular Changes
- functional Changes

15. 15
Role of Osteoblasts in Contribution of SiglecFhigh Lung Neutrophils
KP Tumor accumulation of SiglecFhigh neutrophils depends on Ocn+ cells
Transfer CD45.1+ Lin-c-Kit+
hematopoietic cells in CD45.2+ host
6days
Analysis:
CD45.1+ Progeny

16. Differential Gene Expression
SiglecFhigh neutrophils reveals cancer-promoting phenotypes: Angiogenesis, myeloid cell
recruitment, tumor proliferation cytotoxicity, extracellular matrix remodeling and
immunosuppression
SiglecFhigh Neutrophils Show Tumor-promoting Phenotypes
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17. SiglecFhigh neutrophils reveals cancer-promoting functions: ↑ ROS & ↑ F4/80-expressing cells
(Macrophage)
Tumor-bearing mice Splenic Monocytes and
Myeloid precursors
T-SiglecFhigh; T-SiglecFlow
H-SiglecFlow; CSF-1
Flow Cytometry
SiglecFhigh Neutrophils Show Tumor-promoting Functions
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Ability of Neutrophils to support
tumor promoting myeloid cells

18. Survival plots of lung adenocarcinoma patients
SiglecFhigh Neutrophils are “Not Neutral”
SiglecFhigh neutrophils are tumor-promoting cells: Polarizing towards cancer progression
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19. OR
Quantification of
Osteoblastic Colonies
Tumor-free
(TF)
KP tumor-
bearing (TB)
Serum Factor Contributes to Osteoblast-induced Neutrophils Response
Serum
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Serum components (from tumor-bearing mice) promote osteoblast cells.
Bone marrow cells
ALP: Alkaline
Phosphatase

20. OR
Tumor-free
(TF)
KP tumor-
bearing (TB)
Serum
Compare Protein content
(111 candidates)
sRAGE upregulated in the serum of tumor bearing mice
Protein content in blood of tumor-
bearing mice by protein arrays
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sRAGE was upregulated 2fold in the blood of tumor bearing mice.
sRAGE: soluble Receptor for
Advance Glycation End products
 Member of
Immunoglobulin
superfamily
 Binds Glycoproteins,
Glycolipids & Glycans
C
C
V
 RAGE binds to ligands to trigger pro-
inflammatory cascade
 sRAGE: “Decoy Receptor”
 Binds RAGE ligands & inhibit signal
through the receptor.

21. Quantification of
Osteoblastic Colonies
Tumor-free
Serum ± sRAGE
Bone marrow cells
Role of RAGE in Osteoblastic Activity.
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sRAGE elevated Osteoblasts activity.

22. Neutrophil maturation
(CXCR2 up-regulation)
Tumor-free
Serum
± sRAGE
Hematopoietic stem &
progenitor cells (HSPCs)
± stromal cells
sRAGE increases CXCR2 expression in developing neutrophils
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 sRAGE increased CXCR2 expression
 CXCR2 increased in the presence of Bone marrow Stromal Cells
 Tumor-associated factors can stimulate osteoblastic cells & regulate immune responses.
gMFI: geometric mean fluorescence intensi
Chemokine Receptor-2 (CXCR2) is required for
neutrophil maturation and release into blood stream.

23. Amplification of tumor-
associated neutrophils
Tumor-supportive cells
SiglecFhigh Neutrophil
sRAGE
SiglecFhigh Neutrophils
Activated Ocn+ cells & ↑ activity
Angiogenesis, ECM remodeling
Immunosuppression, cytotoxicity,
Tumor proliferation
Conclusion: A Long-Distance Relay-tionship (LDRt) between Tumor and Bone
Lung tumors “talk” to bone-resident cells to order for “not neutral” neutrophils to foster tumor growth23

24. Discussion & Future Perspective
Limited components (111 factors) were analyzed to investigate the role of tumor in communication with
bone cells.
sRAGE-activated osteoblasts might also directly affect downstream myeloid progenitors. (why only
neutrophils?
There could be other components released by “tumor cells” to communicate with bone-residents cells.
 Elevated sRAGE in plasma is also associated with coronary artery disease, hypertension, the metabolic
syndrome, arthritis and Alzheimer's disease.
Neutrophils phenotypes could be analyzed in sRAGE associated diseases and cancer to understand the
“connection” or rely-tionship.
tEVs could be analyzed to delineate “communication mechanism”
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25. Thank you very much
For
Your Patience!
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