Page 53 - D. Cancer biology

Hes1 in myeloid cells promote immune tolerance through regulation of Arg-1
HyeokGu Kang , Kyung-Hee Chun 1,2
1,2
1 Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, Seoul, Republic of Korea
2 Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Republic of Korea
BACKGROUND
Resistance to tumor treatment can be intrinsic to the tumor cells, but it is often conferred by non-malignant cells that make up the tumor microenvironment (TME). The TME is composed of tissue-resident cells and a large proportion of recruited immune cells that, in certain solid tumor such as breast cancer, can constitute up to 50% of the tumor mass. Macrophages are tissue-resident
innate immune cells important in homeostasis and host defense against pathogens. The biological functions of macrophage are diverse and not only limited to their role as the first line of defense during innate immune response. Macrophages have both tumor-suppressing and tumor-promoting function depending on the TME. It is now generally accepted that tumor associated
macrophages (TAM) have an M2 phenotype and show mostly pro-tumoral functions, promoting tumor cell survival, proliferation, and dissemination. Also support tumor progression by blocking anti-tumor immunity. The immuno-suppressive effect of TAM stems from their enzymatic activities and production of anti-inflammatory mediator, such as arginase-1 (Arg-1), inducible nitric oxide
synthase (iNOS), interleukin-10 (IL-10) and transforming growth factor β (TGF-β), that have an inhibitory effect on tumoricidal lymphocytes. More recently, Franklin RA et al. demonstrated that TAM differentiation requires Notch-dependent transcriptional program. On the other hand, hyper-activation of Notch signaling specifically in macrophage in tumor mass has been shown to suppress
tumor growth in an animal model of cancer. Investigations into how Notch signaling is regulated in TAM and translates into pro- or anti-tumor functions are still not fully understood. Additionally, the downstream molecular mediators of Notch function in TAM are largely unknown.
The Notch signaling pathway regulates cell differentiation, survival, development, and proliferation. In the immune system, Notch signaling is involved multiple steps of B and T cell development, T cell activation, regulatory T cell function, and T helper cell differentiation. Investigation of Notch responses has focused mainly on lymphocytes, and understanding about the effects of Notch
signaling in myeloid lineage cells is more limited. Hes1 belongs to a family of basic helix-loop-helix DNA-binding proteins and is essential for the development of multiple organs and cell types. Consequently, mice that are globally deficient in Hes1 are not viable and show multiple developmental defects. Previous Studies have shown that expression of Hes1 can be regulated by innate and
inflammatory signals. However, the molecular mechanisms, transcriptional targets and physiological relevance of Hes1-mediated modulation of function in macrophage remain largely unknown. According to the preceding circumstances, Notch signaling seems to be important in immune cell development and in functionality. And also there are reports that Notch signaling is a significant
factor in TAM differentiation.
AIM METHODS
1. Heterotopic graft tumor models : 1.0 x 10 5 TC-1 cells, 2.5 x 10 5 B16F10 cells, 5.0 x 10 5 E0771 cells, 5.0 x 10 5 MC-38 cells, 1.0 x 10 6 4T-1 cells and 1.0 x 10 6 CT-26 cells were
In this study, I sought to determine the importance of Notch signaling relative factors in the myeloid compartment of the tumor microenvironment. Especially, injected subcutaneously into the middlelower back of the mice.
Hes1 is highly up-regulated in TAM. And I found that Hes1 deficiency in myeloid cells restricts tumor growth. I observed that Hes1 regulated Arg-1 expression 2. Flow cytometry : Single-cell suspensions were made from the spleens and bone marrow of normal and tumor-bearing mice and tumor tissues. Spleens and bone marrow cells were
of TAM, which is defining feature of immunosuppressive myeloid cells. Therefore, the regulation of myeloid cell function by Hes1 suppressed anti-tumor labeledwith fluorescence-conjugatedantibodies.
function, thereby promoting tumor growth. Taken together, regulation of Hes1 by tumor microenvironment factor plays a role in tumorigenesis and I propose 3. Tumor digestion : Tumors were extracted and finely minced. Tumor tissue was additionally blended with the gentleMACS Dissociator (#130-093-235, Miltenyi Biotec, Berghisch
that Hes1 might be a potential therapeutic target in TAM. Glatbach, Germany) and digested with MACS Miltenyi Tumour Dissociation Kit for mouse (#130-096-730, Miltenyi Biotec) according to manufacturer’s instructions.
RESULTS
Figure 5: Myeloid specific Hes1 knockout mice
Figure 1. Expression of genes encoding canonical notch signaling display normal hematopoiesis. Myeloid-specific Hes1
related factors show different patterns in various types of macrophages.
conditional knockout mice and their littermate controls
(A) Expression of genes encoding canonical notch signaling related factor in display Comparable development of immune systems.
untreated (CTRL) or LPS- or IL-4-treated BMDMs, assessed by next- Six to eight weeks old mice were used. (A-B) Different
generation RNA-sequencing (GSE99296). (B) Gene expression of sorted
population of immune cells were sorted by FACSAria
TAMs and MTMs (mammary tissue macrophages) from 16 weeks old female from tumor tissue and spleen of TC-1 tumor bearing
MMTV-PyMT mice (GES56755). Differentially expressed canonical notch
mice, And then performed a quantitative RT-PCR to
signaling related genes were determined with a p-value threshold of 0.05. Fold check Hes1 mRNA expression level. (C) Cells were
change is depicted using a log scale. sorted from TC-1 tumor bearing mice by FACSAria and
plated. Protein lysates were isolated through cell
homogenization in protein lysis buffer. Protein levels of
Hes1 and Hey1 were detected by western blot. β-actin
was used as a loading control. (D) Total number of Bone
marrow cells, BMDM number and Cell viability after
differentiation from WT and Hes1 deficient littermates
controls. (E) Representative FACS plots of BMDMs in
WT and Hes1 conditional knockout mice. (F-I) Flow
cytometry for stem cells (c-kit + ; Sca-1 + ; c-kit int Sca-1i nt ),
(F) Bone Marrow lymphocytes (T cells: CD3 + ; B cells : B220 + ) and myeloid
cells (neutrophils : CD11b + Gr-1 high ;
monocytes/macrophages : CD11b + Ly-6G dull ) in the bone
marrow; lymphocytes (T cells : CD3 + ; B cells : B220 + ),
myeloid cells (neutrophils : CD11b + Gr-1 high ;
monocytes/macrophages : CD11b + Ly-6G dull ) and T cell
Subpopulation (CD3 + CD4 + T cell; CD3 + CD8 + T cell) in
the spleen.
(G) Spleen
Figure 2. TAMs express higher levels of Hes1 than bone marrow and spleen macrophages. RT and Real-Time PCR analysis of (A) Notch receptor, (B) DLL family, (C)
Jagged family, (D) Hey family and (E) Hes family abundance in bone marrow derived macrophages, spleen derived macrophages and MMTV-PyMT tumor bearing tumor
associated macrophages. The expression level of β-actin was analyzed for normalization control. Data are presented as mean ± SD. (F-G) Mean fluorescence intensity (MFI) of (A) (B) (E) (F)
intracellular Hes1 staining in CD11b + F/80 + double positive macrophages isolated from bone marrow, spleen and tumors (as indicated). (H) Histogram of the intracellular expression
of Hes1. Spleen and tumor cells were subjected to enzymatic digestion each group included at least three mice. Data are presented as mean ± SEM. Significant differences are
indicated by an asterisk (* p<0.05), and p values were calculated using the Student’s t test.
Figure 3. In normal states, Hes1 level is decreased during murine macrophage (C) (D)
differentiation.
(A) Representative flow cytometry plots of CD11b + F4/80 + macrophage cells during murine
bone marrow derived macrophage differentiation at 1,3,5 and 7 days. (B) Graph of
quantifying flow cytometry plot results. (C) Levels of Hes1 gene in BMDM differentiation at
1,3,5 and 7 days by real-time PCR. Data are presented as mean ± SD (n=3). Significant
differences are indicated by an asterisk (* p<0.05), and p values were calculated using the
Student’s t test.
(G) (H) (I) (J) Median survival : 194
200 Days p = 0.0419
5-6
mont p = 0.6573
MMTV-LysM- h Median survival : 215
Hes1 +/+
MMTV-LysM-
Hes1 fl/+ p = 0.0007
Figure 4. Tumor soluble factor and Th2 type cytokine increases Hes1
level, but not hypoxia. BMDMs were exposed to normoxia (20.9% O2; N)
or hypoxia (1% O2; H) for 24 hours. (A) RT and Real-Time PCR analysis
of Hes1 gene. The expression level of β-actin was analyzed for
normalization control (left). Data are expressed as Mean ± SD. Protein
levels of HIF-1α and Hes1 were detected by western blot (Right). Loading
controls were β-actin. (B) In vitro co-culture of BMDMs with or without Figure 6. Myeloid specific Hes1 deletion display tumor growth retardation. (A) Tumor growth in WT and Hes1 deficient littermates subcutaneously injected with B16F10
melanoma cells (LysM-Hes1 +/+ n=3, LysM-Hes1 fl/fl n=3 mice). (B) Tumor growth in WT and deficient littermates subcutaneously injected with E0771 breast cancer cells (Cre -
tumor cell line and human embryonic kidney cell line. Protein levels of
NICD, Hes1, p-STAT3 (Y705), STAT3, PD-L1, Hey1, Arg-1, iNOS, CD206, ,Hes1 fl/fl n=5, Cre + ,Hes1 fl/fl n=5 mice). (C) Tumor growth in WT and deficient littermates subcutaneously injected with MC-38 colon carcinoma cells (Cre - ,Hes1 fl/fl n=5, Cre + ,Hes1 fl/fl
n=5 mice). (D) Tumor growth in WT and deficient littermates subcutaneously injected with TC-1 Lung adenocarcinoma cells (Cre - ,Hes1 fl/fl n=5, Cre + ,Hes1 fl/fl n=5 mice). (E) Tumor
p-Erk1/2 (Thr202/Tyr204) and Erk1/2 were detected by western blot. weight of B16F10, E0771, MC-38 and TC-1 cancer cells implanted into the WT and deficient littermates. Color scheme represents WT (black), Hes1-deficient (red) mice. Each
Erk1/2 was used as loading control. (C) BMDMs were exposed or not to
murine recombinant IL-4 (20 ng/mL), IL-10 (10 ng/mL), IL-13 (10 ng/mL) group included 3-5 mice. (F) Tumor growth in mice treated with anti M-CSF-1R (CSF1R antibody), anti Ly-6G (Ly-6G antibody) and anti IgG. Data are expressed as mean ± SEM.
Each group included three mice. Significant differences are indicated by an asterisk (* p<0.05), and p values were calculated using the Student’s t test. Myeloid-Hes1 KO PyMT
alone or in combination for 36 hours. Protein levels of Hes1, NICD, IRF-4,
PPAR-γ, and STAT3, p-STAT6 (Y641), STAT6, Arg-1, CD206 and tumors were smaller than WT PyMT mice. (G) At 200days, tumors were isolated and presented in a photograph. (H) Total tumor burden of LysMcre-Hes1 +/+ PyMT and LysMcre-
Hes1 fl/+ PyMT mice at 20 and 24 weeks of age (n=17) and (I) Tumor multiplicity in a statistical graph. (J) Survival of mice tumor bearing of LysMcre-Hes1 +/+ PyMT (N=10) and
GAPDH were detected by western blot. GAPDH was used as loading LysMcre-Hes1 fl/+ PyMT mice (N=14). LysMcre- Hes1 fl/+ PyMT mice display prolongedsurvival. Data are mean ± SD.
control.
(A) (B)
CONCLUSION REFERENCES ACKNOWLEDGEMENTS
• In the TAM, Hes1 is higher level than bone 1. [Quail DF, Joyce JA. Microenvironmental This work was supported by
marrow derived macrophage or spleen regulation of tumor progression and metastasis. the Basic Science Research
derived macrophage. Nature medicine 2013;19(11):1423-1437] Program through the National
Research Foundation of
• Hes1 is increased by Th2 type cytokines and 2. [Engblom C, Pfirschke C, Pittet MJ. The role of Korea (NRF) funded by the
tumor soluble factor, but not hypoxia, which myeloid cells in cancer therapies. Nature reviews. Ministry of Education (NRF-
known as typical characteristics of TME. Cancer 2016;16(7):447-462] 2020R1IA1A0107289311).
• In mice, engineered to selectively lack 3. [Pollard JW. Tumour-educated macrophages (C) (D)
myeloid cell Hes1, display retardation of tumor promote tumour progression and metastasis.
Nature Reviews Cancer 2004;4(1):71-78]
growth and prolonged mouse survival. The Contact
ratio of CD45+ cells and myeloid cells in the 4. [Franklin RA, Liao W, Sarkar A, Kim MV, Bivona
tumor tissue was no difference in both WT MR, Liu K, et al. The cellular and molecular origin information
and cKO mice. of tumor-associated macrophages. Science
2014;344(6186):921-925] Address : Department of Biochemistry
• Ablation of Hes1 in myeloid cells decreased & Molecular Biology, Yonsei University
intracellular expression of the anti- 5. [Tanigaki K, Honjo T. Regulation of lymphocyte College of Medicine, 50-1 Yonsei-ro,
inflammatory marker Arg-1 in TAM. development by Notch signaling. Nature Seodaemun-gu, Seoul 03722, Figure 7. Hes1 regulates TAM functions. (A-B) Percentage of total, monocyte, granulocyte, DC, macrophage, T, B and NK cells within the CD45+ population of tumor infiltrating
Repubic of Korea
immunology 2007;8(5):451-456] cells isolated from TC-1 tumors engrafted in LysM-Hes1 +/+ and Lysm-Hes1 fl/fl mouse, estimated by flow cytometry. Data are expressed as individual data points (n>5 mice per
• Taken together, regulation of Hes1 in TAM by Telephone (Mobile) : +82-10-5331- genotype as shown) and mean bar ± SEM. (C) Mean Fluorescence Intensity of CD206, IFN-γ, IL-12 and Arg-1 expression on the TAM isolated from TC-1 tumor engrafted into the
TME plays an important role in tumorigenesis 6. [Osborne BA, Minter LM. Notch signalling during 9448 indicated WT and Hes1 deficient littermates (n=5 per group), measured by flow cytometry. Data are expressed as mean ± SEM. (D) Quantitative PCR of metabolic enzyme (Arg-1,
and I suggest that Hes1 might be a potential peripheral T-cell activation and differentiation. iNOS), cytokines (IL-6, IL-10, IL-12p40, IFN-γ, TNF-α, VEGF, MMP-9), immune checkpoint (PD-L1, PD-L2) and co-stimulatory molecules (CD80, CD86) genes in isolated Hes1
therapeutic target in TAM. Nature reviews Immunology 2007;7(1):64-75] Email : Hk1038@yuhs.ac, deficient TAM relative to control littermates. Data are presented as mean ± SD. (E-F) Percentage of M-MDSC and G-MDSC in bone marrow and spleen. Data are expressed as
time_liberty@hanmail.net mean ± SEM. Significant differences are indicatedby an asterisk (* p<0.05), and p values were calculated using the Student’s t test.