Targeting Fusion Machinery Between Macrophages and Cancer Cells to Ameliorate Cancer Desmoplasia
Conditions: Fusion Protein Expression, Bone Marrow Cells
Sponsor: National Taiwan University Hospital

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版本日期：20220310 v3 
國立臺灣大學醫學院附設醫院 
研究計畫書 
□藥品  □醫療器材  □醫療技術  V 其他 
一、計畫名稱： 
中文: 標定巨噬細胞與癌細胞融合機制以改善癌症纖維增生 
英文：Targeting fusion machinery between macrophages and cancer cells to 
ameliorate cancer desmoplasia 
二、主要主持人：姚明醫師 
三、協同主持人：羅仕錡醫師、常慧如醫師 
四、摘要 
臨床及臨床前研究顯示, 癌細胞能與血球細胞融合, 而獲得移動, 增生, 與藥物抗性的特
性. 我們以往在小鼠模式研究, 發現腸道間質細胞與巨噬細胞融合, 導致慢性腸道纖維化. 我
們認為, 與巨噬細胞之融合癌細胞, 可能導致腫瘤纖維化與惡化. 
為研究與血球細胞融合癌細胞, 我們以cre-loxP 系統, 或性別錯配方式, 建立小鼠腫瘤模
型; 也建立由ROSA mice 骨髓, 移植給予pdx-cre-Kras LSL-G12D (KC) mice 的研究模型. 融合
細胞與巨噬細胞生物標記, 以免疫螢光染色和流式細胞儀偵測.  
在實驗室中, 我們以cre-loxP 系統, 或雙螢光染色方式, 評估胰臟癌細胞與巨噬細胞, 共
培養後的細胞融合現象. 我們將會檢測融合細胞的增生, 移動, 與藥物抗性. 融合與未融合的
癌細胞基因型態, 將以Affymetrix 微陣列分析. 
在小鼠腫瘤模式中, 胰臟癌細胞與宿主小鼠細胞融合機率, 在腫瘤植入1 到5 周後, 為1.6 
to 4.86%. 在KC 小鼠模型, 於骨髓移植後7-16 周, 融合細胞比率為0.62 to 26.49%. 以脂質體氯
磷酸鹽去除巨噬細胞, 小鼠的腫瘤生長減緩, 細胞融合比率降低. Masson 三色染色, 與免疫印
跡實驗顯示, 腫瘤纖維化狀態與細胞融合程度相關. 
在實驗室中共培養巨噬細胞與胰臟癌細胞, 顯示24 到72 小時內細胞融合比率0.64 to 
1.12%. 在小鼠骨髓細胞中去除CD11b(+)細胞,明顯降低與癌細胞細胞融合比率. 融合癌細胞較
野生胰臟癌細胞, 顯示較高的移行, 增生能力; 以及對放射照射的抗性. 基因分析顯示
degradation of the extracellular matrix, 以及signal regulatory protein (SIRP)訊息傳遞路徑,在融合
細胞,較未融合細胞明顯上升. 核糖核酸與蛋白測量,顯示細胞外基質,纖維化,及 SIRP 分子表
現量, 在融合細胞較高. 
綜合言之,與巨噬細胞融合的胰臟癌細胞,與腫瘤纖維化以及癌症惡化相關.SIRP 訊息傳遞
路徑, 是具有潛力破壞細胞融合, 改善胰臟癌纖維化與惡化的標的. 
我們將收集以往接受骨髓移植之後, 發生惡性腫瘤的病理組織.評估其癌細胞中, 是否含
有來自捐髓者的基因.如:Y 染色體,或短串聯重複(short tandem repeats) 研究… 以證明臨
床上, 癌細胞與巨噬細胞融合的現象.預計由病理部,收集20 位符合條件病患之腫瘤檢體切片,
每位10 片各4-8m. 

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另將收集健康人,或多血症病患之周邊單核球細胞,作為實驗室中,與癌細胞株共培養,篩
選純化融合癌細胞, 作為對於融合癌細胞之增生,輻射敏感性,轉移能力… 等等生物特性研究.
預計由檢醫部血庫,收集30 位健康捐髓者或多血症病患之周邊血液單核球細胞,一次10-20ml.  
 
Abstract  
Clinical and preclinical evidence reveal that cancer cells may fuse with hematopoietic cells to 
obtain properties including migration, proliferation and drug resistance. Our previous studies in 
murine model revealed fusion hybrids between intestine stromal cells and macrophages contribute 
to chronic intestine fibrosis. We hypothesize that cancer cell-macrophage fusion hybrids may lead to 
pancreatic cancer desmoplasia and progression.  
 
Murine tumor models using cre-loxP or gender-mismatched xenografts as well as 
pdx-cre-KrasLSL-G12D mice after bone marrow transplantation from reporter ROSA mice were 
established. Fusion hybrids and macrophage markers were detected using immunofluorescence 
staining and flowcytometry. 
 
In vitro co-culture using cre-loxP or dual fluorescence methods of pancreatic cancer cells with 
macrophages was used to evaluate the frequency of fusion phenomenon. The proliferative, 
migratory and resistant phenotypes of purified fusion hybrids were measured. Differentially 
expressed genes between fusion hybrids and non-fused cancer cells were compared by Affymetrix 
microarray analysis.  
    Fusion between pancreatic cancer and host derived cells in murine xenograft model ranges 
from 1.6 to 4.86%, 1-5 weeks after tumor implantation. In KC murine model, fusion hybrids of 0.62 
to 26.49% was noted 7-16 weeks after BM transplantation from ROSA mice. Depleting macrophage 
using liposomal clodronate in mice with tumor xenograft showed delayed tumor growth and 
reduced fusion hybrids. Masson trichrome stain and immunoblots revealed the fibrosis status of 
tumors were correlated with the amount of fusion hybrids.   
In vitro co-culture of macrophages with pancreatic cancer cells showed spontaneous fusion of 
0.64 to 1.12% in 24 to 72 hrs. Depleting CD11b(+) cells from BM cells decreased the level of fusion 
phenomenon significantly. Fusion hybrids revealed enhanced migratory and proliferative ability as 
well as resistance to radiation cytotoxicity compared with wild type pancreatic cancer cells. Gene 
set enrichment analysis showed the degradation of the extracellular matrix (ECM) and signal 
regulatory protein (SIRP) signal pathway were significantly upregulated in fusion hybrids compared 
with non-fused pancreatic cancer cells. The expression level of ECM, fibrosis, SIRP molecules were 
elevated at RNA and protein levels in fusion hybrids.  
In summary, fusion between pancreatic cancer cells and macrophages contribute to cancer 
desmoplasia and malignant progression of pancreatic cancer. SIRP signal pathway is a potential 
target to disrupt fusion phenomenon and ameliorate pancreatic cancer fibrosis and progression.  
 
We are going to collect tumor tissues from cancer patients who received allograftic bone 
marrow transplantation before. We will evaluate Y chromosome or short tandem repeats to 

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identify donor- derived genes in cancer cells and demonstrate the clinical evidence of fusion 
between cancer cells and macrophages. The tumor tissues will be collected from the Pathology 
Department. Ten slides of 4-8m will be collected from twenty patients enrolled according to the 
inclusion criteria. 
 
We will collect peripheral mononuclear cells from healthy volunteer ( eg. Donors for bone 
marrow transplantation) or hyperemia patients. The mononuclear cells will be induced to 
differentiate into macrophages and will be co-cultured with cancer cells in order to purify fusion 
hybrids. The fusion hybrids between cancer cells and macrophages will be evaluated for biologic 
characters including proliferation, radio-sensitivity, migration etc. We planned to collect blood 
samples from Department of Laboratory Medicine, Blood bank. Thirty subjects of healthy volunteer 
or hyperemia patients will be enrolled. Ten to 20ml peripheral blood will be collected from each 
subjects for one time. 
 
五、研究主題說明 
癌症纖維增生, 尤其在胰臟癌, 是導致癌症惡化, 對治療產生抗藥性的主要原因
之一. 目前針對剔除被癌症活化之纖維細胞的治療, 效果不如預期, 反而會導致更具
侵略性的癌症, 吸引抑制免疫反應的T 細胞, 大量湧入腫瘤微環境. 
考量巨噬細胞在發炎反應, 纖維化, 癌症發生, 與惡化的角色, 許多針對巨噬細
胞剔除或極化的治療發展, 得到具有潛力的臨床前期結果. 研究顯示巨噬細胞與癌細
胞的細胞融合, 是導致腫瘤多樣性的動力, 引起抗藥性與癌症惡化. 但對於巨噬細胞
與癌細胞的細胞融合, 在癌症纖維增生的研究很少, 為改善胰臟癌治療效果, 提高醫療
經費經濟效益與病患生活品質, 我們將發展以巨噬細胞為主的治療策略, 改善癌症纖維
化環境, 達到預防腫瘤新生、惡化, 以及提高治療效果的目的. 
我們將前瞻性收集健康受試者之周邊單核血球, 在實驗室中分化為巨噬細胞, 再
與癌細胞共培養, 挑選出與巨噬細胞融合的癌細胞, 進行細胞與動物實驗, 檢測融合細
胞之生物特性, 包括纖維化激素分泌.  
另外將回溯性收集接受骨髓移植後, 罹患惡性腫瘤的病患組織, 檢測腫瘤中是否
含有來自捐髓者的基因, 包括巨噬細胞特性基因, 以及與巨噬細胞融合的癌細胞表現量, 
並與腫瘤纖維化程度進行比對. 
 
六 研究目的及背景說明（包括學理根據及有關文獻報告） 
Desmoplasia has been shown to exert mechanical forces and create a biochemical 
intra-tumoral immunity and influence the development and progression of a malignancy.1 Recent 
reports suggest a significant negative correlation between cancer patient survival and extracellular 
matrix deposition in primary tumors; and concomitant stromal targeting may enhance therapeutic 
outcomes in cancer patients.2  
In defining the source of the dense stroma, it is generally believed that over-activated CAFs 

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could produce extracellular matrix (ECM) proteins, the major component of the dense stroma. 
Anti-fibrosis drugs including those depleting CAFs or collagen, sonic hedgehog inhibitor, antibodies 
against LOX/LOX2, were under clinical trials.1 However, CAF-depleted tumors displayed a more 
aggressive phenotype and alteration in regulatory T cells, raising the possibility that CAF-targeting 
strategies may have both beneficial and detrimental effects.2  
Considering the functional significance of macrophages in inflammation, desmoplasia, cancer 
initiation and progression, mounting studies had conducted to evaluate the efficiency of 
anti-macrophage as a novel strategy against cancer.3 Accordingly, the anti-macrophage strategies 
include the inhibition of the monocytes recruitment as well as transformation, and the ablation the 
macrophages directly. Given the multifaceted roles of maintaining homeostasis, the systemic 
depletion of macrophages may lead to increased infections or impaired ability of tissue-resident 
cells to carry out their normal function. Thus, the identification of tumor associated macrophage 
(TAM)-specific markers will enable the development of more sophisticated therapies that can be 
targeted specifically to tumors without affecting the function of other tissue-resident immune 
cells.4  
Recent studies had identified numerous factors such as platelet-derived growth factor (PDGF) 
and transforming growth factor 1 (TGF1) released by TAMs, especially bone marrow 
(BM)-derived ones,5 involving in the process of activating CAFs. In addition, there were 
TAMs-derived pro-fibrotic factors, such as connective tissue growth factor (CTGF), chemokine 
ligand 17 (CCL17), CCL22 and reactive oxygen species (ROS). Similar observation was found in 
TAMs-derived matrix metallopeptidase 9 (MMP9) which involved in the stroma turnover by 
degrading the ECM proteins.6 However, rare study investigates the role of fusion between 
macrophages and cancer cells in desmoplasia and tumor progression as well as the strategy 
targeting fusion machinery of macrophages with cancer cells. 
Several lines of evidence support that cell fusion between cancer cells and leukocytes, majorly 
macrophages, is one of the explanations for tumor resistance and progression.7 Recent genotyping 
of a metastatic melanoma to the brain that arose following allogeneic BM transplantation supports, 
first time clinically, fusion between a BM derived cell and a tumor cell playing a role in the origin of 
metastasis. Fusion hybrids enumerated in peripheral blood of pancreatic cancer patients were 
reported to correlate with disease stage and predict overall survival.8 Furthermore, general 
inflammatory responses were demonstrated to increase the number of fusion events.9 While the 
fusion events were demonstrated in animal and human, and were correlated with cancer 
resistance and metastasis,7 little is known regarding key questions such as the mechanisms through 
which macrophage-cancer cell fusion and subsequent genomic hybridization occurs in vivo; 
potential survival advantages of hybrids; reprogramming of fibrotic and immunologic tumor 
microenvironment by hybrids; and the development of novel strategies to target fusion machinery. 
The majority of macrophages, including tissue macrophages in normal tissue and TAMs, are 
derived from bone marrow (BM).5 Our preliminary study, using irradiated murine intestine model, 

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suggested parallel between the development of cell fusion between BM-derived monocytes and 
intestine stromal cells with radiation-induced fibrosis.10 Using cre-loxP system and gender 
mismatched BM transplantation, we demonstrated that BM-derived CD11b(+) myelo-monocytic 
cells/macrophages were the major fusion partner to damaged intestine stromal cells after radiation. 
The fusion events in irradiated intestine stroma correlated with chronic intestine fibrosis with 
enhanced TGF transcripts and collagen deposition. With macrophage deletion by liposomal 
chlodronate or conditional CD11b knock-out, we can ameliorate radiation induced chronic intestine 
fibrosis.10 
The main property of cancer cells that makes them malignant is the ability to produce diverse 
progeny. Fusion between cancer cells and TAMs can be an engine of genomic and epigenetic 
variability that has a potential to make cells with new properties at a rate exceeding that achievable 
by random mutation.11 Previous reports revealed cell fusion contribute to drug resistance and can 
promote the ability of cancer cells to metastasize.7  
Fibrosis and inflammation are hallmarks of tumor desmoplasia. TAMs, majorly derived from 
bone marrow,5 act as an essential connecting moiety between inflammation and cancer via 
secretion of pro-inflammatory cytokines/chemokines. The M2 polarized macrophages, which 
constitute majority of the macrophages in tumor microenvironment, secrete IL10 and other 
cytokines that mediate T helper 2 (Th-2) responses and are responsible for malignant tumor 
tansformation and inhibit antitumor immune response mediated by T cells. TAMs were reported to 
play a phenomenal role in enhancing mesenchymal phenotype of cancer cells via TGF. The 
lipopolysaccharide (LPS) receptor toll-like receptor 4 (TLR4) on the surface of TAMs has been 
implicated in a role in the EMT via TLR4/Interleukin 10 (IL10) cascade.12 The level of secreted protein 
acidic and cysteine-rich (SPARC) gene, a regulator of EMT, was reported to be 3-4 fold higher in 
hybrids of BM-derived cell and melanoma.13 
Analysis of microenvironment microarray assay revealed that fusion hybrids of colon cancer 
cells and macrophages exhibited a combination of biases, reflecting properties of both parental 
cells, including fibronectin, collagen XXIII, vitronection, potentially providing a broader 
desmoplastic process in different microenvironment.8 Our preliminary results of differential 
expression of genes between fusion hybrids of cancer cells and macrophages with non-fused 
cancer cells show significant elevation of signal pathways involving inflammation, extracellular 
matrix degradation and mesenchymal phenotypes etc. Besides, prominent increase in signal 
regulatory protein (SIRP) family members, a cell-cell communicating system, were also found.  
Current treatment result of cancer with desmoplasia is unsatisfactory. It is generally believed 
that desmoplastic stroma provides cancer cells with cytokines/chemokines to suppress immune 
surveillance and to enhance malignant progression. Recent clinical trials revealed direct targeting 
desmplasia using sonic hedgehog inhibitor lead to a more aggressive phenotype with increased 
regulatory T cells.1,14 On the other hand, depleting macrophage or its recruitment from circulation 
revealed to be effective in reducing pro-fibrotic cytokines/chemokines and CAFs activity; however, 

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with significant normal tissue toxicity.4  
From previous investigation and our study, fusion hybrids between cancer cells and 
macrophages appeared to play important role in promoting cancer desmplasia as well as malignant 
progression.8 We proposed to develop an alternative method to ameliorate cancer desmoplasia by 
focusing on novel targets interfering fusion between cancer cells and macrophages. The potential 
candidates were revealed by our preliminary microarray study. eg. Signal regulatory protein (SIRP) 
signal pathway including SIRP, a macrophage fusion receptor,15 which was nearly 5 folds 
differentially expressed in fusion hybrids. Furthermore, the down-stream molecules including 
DAP12 (DNAX activating protein), a general macrophage fusion regulator, and protein tyrosine 
phosphatase (PTPN6), protein tyrosine kinase (PTK2B) were increased 8 folds and 4 folds, 
respectively, in fusion hybrids.  
We proposed to target on SIRPand other potential candidate molecules with neutralizing 
antibodies or silencing mRNA to reduce the incidence of fusion phenomenon in cancer with 
desmoplasia. By reducing the frequency of tumor heterogeneity and the fibrotic phenotype, we 
would like to improve the therapeutic outcome of cancer with desmoplasia. The combination effect 
of anti-fusion agents with other anti-fibrosis agents or immunologic therapy will also be explored 
 
七 研究方法與程序 
(1) 
To demonstrate tumor fibrosis and fusion between bone marrow derived 
monocytes and cancer cells in human specimen and in murine model. 
Murin model 
pdx-Cre-Kras-mutated (KC) mice of 10~12 wk-old, a spontaneous pancreatic 
adenocarcinoma murine model, will be given whole body irradiation followed by bone 
marrow transplantation from ROSA mice (Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo/JNarl). 
U937(fl-dsRed-fl-eGFP). Mice will be sacrificed at 16~26wk-old. Pancreas tissues will be 
evaluated for fibrosis status using masson trichrome stain, immunoblots and qPCR 
measurement of fibrosis and extracellular matrix proteins. Fusion between pancreatic 
cancer cells with bone marrow derived cells will be identified as cells expressing green 
fluorescence due to DNA recombination. Fusion hybrids will be quantified and further 
immune-stained with macrophage, lymphocyte markers.   
Human specimen 
Tumor tissue specimens of patients receiving bone marrow transplantation from 
mismatched gender (eg. female receiving bone marrow cells from male donor) in 
previous will be collected. Cancer cells will be evaluated with Y chromosome expression 
using fluorescence in situ hybridization. Tumor fibrosis score will be measured after 
Masson trichrome stain.  
(2) 
To evaluate the functions and the expression of fibrosis, and inflammatory 
molecules after fusion between cancer cells and macrophages in vitro.  

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Cell culture 
Cancer cells including Pan02 (mouse pancreatic epithelial cancer cells) and Panc-1 
(human pancreatic adenocarcinoma cells) were cultured in Dulbecco’a modified Eagle’s 
medium (DMEM) + 10% serum. Stable cancer cell lines, Pan02(Cre), Pan02(H2B-YFP/Cre), 
Panc-1-EGFP were generated by retroviral transduction and polyclonal populations were 
selected by antibiotic resistance and flow-sorted for bright fluorescence as appropriate. 
Primary macrophages derivation was conducted from the bone marrow of ROSA mice. 
U937-loxP-dsRed-loxP 
cells 
were 
generated 
by 
stably 
expressing 
a 
MSCVLPdsRed-LoxP-eGFP-PURO construct (Addgene#32702) into the parental U937 
cells. Primary monocytes derived from healthy peripheral blood donors will be 
collected after CD14+ magnetic beads selection. To elicit macrophages, cells were 
cultured for 6 days in DMEM + 15% serum supplemented with sodium pyruvate, 
nonessential amino acids and CSF1(25ng/ml). 25ng/ml GM-CSF or 50ng/ml M-CSF was 
used to promote M1- or M2-like polarization.   
Co-culture to generate fusion hybrids 
Cell 
fusion 
hybrid 
generating 
co-cultures 
were 
established 
in 
macrophage-derivation media without CSF1 for 4 days. Pancreatic cancer cells and 
macrophages with stably expressed fluorescence or labelled with fluorescent dyes 
(CytoTellTM) were co-seeded at a 1:2 ratio at low density. Hybrid cells were FACS-isolated 
for appropriate fusion markers on a FACS sorters. Low-passage hybrid isolates were 
established; functional experiments were conducted on 8 to 20 hybrid isolates. Live 
imaging of co-cultured cells was performed using an automated microscope system and 
associated software.  
Invasion analysis 
Cellular invasion was measured in a growth factor-reduced Matrigel invasion 
chamber with 8 m pores. Cells (3x105) in a medium containing 0.1% FBS were placed 
into each Boyden chamber. The medium containing 10% FBS was placed in the lower 
chamber to facilitate chemotaxis. Invasion assays were run for 15 hours, and then cells 
that passed through the Matrigel membrane were stained with 0.09% crystal violet/10% 
ethanol. After extraction by elution buffer, the stain was measured at 560nm. 
Representative images of invaded cells were taken by microscope.  
Proliferation assay 
A suspension of hybrid or wild type cancer cells in DMEM+4% FBS was added to 
each well of 96-well plates. Plates were cultured in a humidified incubator for 72 hours, 
after which 5 l of MTS reagent was added to each well. Two hours later, absorbance at 
490nm was read with a plate reader.  
Immunoblots 
Hybrid or wild type cancer cells were collected and lysed in protein lysis buffer 

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containing a protease inhibitor cocktail. The supernatants were centrifuged at 4oC for 15 
min at 13000rpm. Twenty micrograms of total protein were loaded into each well of a 
sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and separated. Protein 
was transferred onto a polyvinylidene fluoride membrane, and membranes were 
incubated in blocking solution at room temperature for 1 hr, followed by incubation 
with the indicated primary antibodies (antibodies to fibronectin, TGF1, collagens, 
Interleukin 1 , etc) at 4oC overnight. The membranes were washed in TBS-T and 
incubated at room temperature with peroxidase-conjugated secondary antibodies for 1 
hr. Signals were detected using the enhanced chemiluminescence assay.  
(3) 
To demonstrate the development of desmoplasia and fusion hybrids between 
cancer cells and bone marrow-derived macrophages during cancer progression in 
murine model 
Mice and bone marrow transplantation 
The following strains were used in the described studies: C57BL/6J mice, ROSA 
mice [Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/JNarl], and KC mice [KRASmut/+/Cretg/0], Kras 
mice [KRAS mut/+]. For tumor growth, 8 to 12 week-old C57BL/6J mice were injected with 
5x104 cells subcutaneously. Length and width of palpable tumors were measured three 
times weekly with calipers until tumors reached a maximum diameter of 2 cm. Tumors 
were surgically removed and animals were sacrificed during tumor removal. For growth 
of tumor at metastatic sites, 1x106 cells were injected into the spleen. Livers were 
analyzed 3 weeks later for tumor burden by H&E stain.  
To evaluate fusion hybrids during tumorigenesis, two sets of experiment were 
conducted. (1) ROSA mice at 8 to 10 week-old were subcutaneously injected with 5x104 
cancer cells stably with or without expressing cre recombinase. (2) KC or Kras mice were 
irradiated at 8 to 10 week-old followed, within 24 hr, by bone marrow transplantation 
(BMT) from ROSA mice. Mice were sacrificed at various time points after BMT to 
evaluate pancreatic tissues. H&E and immunofluorescence stain were performed to 
measure the level of pancreatic malignancy and fusion hybrids.  
Immunofluorescence analysis 
Primary tumors or livers were fixed in 10% buffered formalin, frozen in optimum 
cutting temperature (OCT), and 4m sections were obtained. Tumors were incubated 
with antibodies for GFP followed by detection with fluorescent secondary antibody. 
Nuclei were counterstained with Hoechst. Slides were digitally scanned and analyzed. 
Confocal images were acquired with an Olympus confocal microscope.  
Tumors from KC or Kras mice were fixed in 4% paraformaldehyde for 2 hours at 
20oC, washed and cryopreserved in 30% sucrose for 16 hr at 4oC and then embedded in 
OCT. Sections were cut to 4m thickness, baked for 30 min at 37oC, then subjected to 
antigen retrieval under standard conditions, blocked with DAKO protein block 

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serum-free, and incubated for 16 hr at 4oC with primary antibodies, anti-dsRed, anti-YFP, 
and anti-GFP in background-reducing antibody diluent. Fluorescent-tagged secondary 
antibodies were applied, and then sections were mounted in a ProLong Gold antifade 
reagent. Antibody specificity was determined by immunostaining healthy livers of 
non-tumor bearing mice and performing secondary antibody only controls. 
(4) 
To correlate desmoplasia with fusion hybrids during cancer progression by 
macrophage depletion in murine model 
Pravastatin, and clodronate treatment 
To suppress cancer desmoplasia, mice were given pravastatin, a Rho pathway 
inhibitor, 30mg/kg/day in drinking water for 10 weeks starting two weeks before tumor 
implantation. To deplete macrophages, Liposomal Clodronate 2.5mg/ml in PBS 
100L/mouse, was injected once every three days for two weeks before sacrifice.  
Immunohistochemical analysis 
Sections were stained with hematoxylin-eosin as well as Masson Goldner trichrome 
staining to evaluate collagen deposition. Fusion hybrids were measured by 
immunofluorescence staining at serial histology section and correlated with Masson 
trichrome staining.   
(5) To validate in vivo-derived cell fusion hybrids 
For 
demonstration 
that 
tumor 
cells 
can 
fuse 
with 
myeloid 
cells, 
Pan02(fl-dsRed-fl-eGFP) cells were injected intradermally into 8 to 10 wk-old Cd11b-DTR 
mice [B6.FVB-Tg(ITGAM-DTR/EGFP)34Lan/J]. Mice were given diphtheria toxin was given 
10ng/g i.p. every three days for two weeks before sacrifice. When tumors reached 1cm3, 
primary tumors were removed for immunofluorescence analysis. 
Tumors were diced and digested for 30min at 37oC in DMEM + collagenase 
A(2mg/ml) + DNase under stirring conditions. Digested tumor cells were filtered 
through a 40m filter and washed with PBS. Cells were washed and resuspended in 
FACS buffer. Cells were incubated in PBS containing LIVE/DEAD Fixable Aqua with Fc 
Receptor binding inhibitor. Cells were then incubated in FACS buffer for 30 min with 
fluorescence labelled primary antibodies including GFP, DsRed, cytokeratin, CD45, CSF1R, 
F4/80.  FACS machine was used for analyses. The level of fusion hybrids expressing 
macrophage markers, unfused cancer cells were evaluated.    
(6) To conduct karyotype and gene expression analysis of fusion hybrids 
Karyotype analyses 
Chromosome spreads from cells in S phase were prepared from cells treated for 
>12 hr with colcemid (100ng/ml) to induce mitotic arrest. DNA was visualized by 
staining with DAPI. Images of stained fixed cells and chromosome spreads were 
acquired by inverted microscope. Chromosomes were counted manually. A minimum of 
20 cells were analyzed in each experiment.  

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Gene expression analysis 
Microarray analysis was performed with gene chips of Affymetrix and data were 
analyzed using software to identify relative expression differences between cell types 
(wild type cancer cells, macrophages, non-fused cancer cells or fusion hybrids) and 
produce gene ontology (GO) analyses. GO category enrichment was calculated using the 
GOstats R package and visualized using functions from the GOplot R package.  
(7) To validate the differential expressed signal molecules between fusion hybrids and 
non-fused cancer cells. 
Polymerase chain reaction 
DNA was extracted from wild type cancer cells and fusion hybrids and incubated 
in lysis buffer followed by neutralization with neutralization buffer. Primers of forward 
and reverse sequences were prepared according to the candidate molecules (eg SIRP, 
DAP12 etc.). Reactions were run with a 60oC annealing temperature.  
Immunoblots 
As described previously. The primary antibodies will be candidate signal 
molecules of fusion machinery eg. SIRP, DAP12 etc. 
(8) To evaluate the efficacy of small molecules interfering fusion machinery in 
suppressing fibrosis development in murine model.  
In vitro fusion assay to evaluate anti-fusion agents 
Freshly isolated macrophages from ROSA mice or U937(fl-dsRed-fl-eGFP) were 
co-cultured with Pan02(H2B-YFP/Cre) or Panc-1-EGFP cells supplemented with 
anti-SIRPneutralizing antibody, PTK2B inhibitor at various concentrations. The cells 
were examined daily for 4 days. The level of fusion was graded by FACS of GFP. 
Expression of fibrosis related molecules including TGF, fibronectin were measured by 
immunoblots and PCR described previously. 
Murine model to evaluate anti-fusion agents 
Mice were injected i.p. with normal IgG, anti-SIRP antibody three times a week 
beginning immediately after the establishment of murine models of primary tumor and 
liver metastatic tumor described previously. Tumors were measured with digital calipers 
and tumor volume was calculated. Mice were sacrificed to obtain primary and 
metastatic tumors for immunofluorescence study to detect the level of fusion hybrids. 
Tumor tissues were prepared for immunoblots and PCR to evaluate fibrosis status.  
(9) To evaluate the combination of fusion interfering agents and other agents in 
ameliorating cancer progression. 
Pan02(H2B-YFP/Cre) or Panc-1-EGFP cells were injected subcutaneously into the 
flanks of 8 week-old ROSA mice. The mice were injected i.p. with normal IgG or 
anti-SIRP, each with or without anti-PD1 antibody, twice a week beginning immediately 
after tumor implantation. Tumor growth were measured. Mice were sacrificed to 

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evaluate the level of fusion hybrids and fibrosis as described previously. The combination 
of anti-SIRPwith chemotherapy, eg. Gemcitabine, will be explored as well. 
 
八 受試者選擇標準 (檢體提供者)： 
預計收案人數：50 人 
身分：█病人：█健檢人：█其他：  捐髓, 放血者…           
類別：前瞻性收集周邊單核血球: 30 人; 健檢, 捐髓, 放血者…  
      回溯性收集惡性腫瘤組織: 20 人; 惡性腫瘤且曾接受骨髓移植之病患.  
 
九、研究設計與流程（與臨床研究相關部份）： 
1. 前瞻性收集周邊單核血球 
由受試者周邊血管抽血, 收集約10-20ml, 置於添加抗凝血劑試管中, 再進行後續處理. 
納入條件(參加本研究的條件):  
(1) 於進案一周內,有正常的周邊血液檢測結果. 
(2) 若為捐髓者,可以為接受血球刺激或生長激素之後.  
(3) 可以為血紅素偏高的放血者. 
(4) 年齡 20-70歲. 
排除條件(若您有下列任一情況，您將無法參加本研究):  
(1)有惡性腫瘤,且無復發狀態少於五年者. 
(2)有重大心血管疾病, 免疫疾病, 懷孕.  
(3)長期服用免疫抑制劑或類固醇者. 
 
2. 回溯性收集惡性腫瘤組織 
納入條件 
(1) 曾接受骨髓移植, 且骨髓移植完成之後發生固態腫瘤. 
(2) 曾接受切片或手術切除腫瘤, 檢體儲存於病理部之剩餘腫瘤組織, 約4-8m 厚, 
10 片. 
排除條件 
(1)無. 
 
十、計畫預定進度： 
自研究倫理委員會核准後，開始收集檢體3 年, 包括前瞻收集周邊血液30 名, 回溯
收集腫瘤組織20 名. 檢體將保存10 年(或檢體保存至2032 年)。 
 
十一、研究對象權益之保障、同意之方式及內容： 
於門診或病房進行口頭告知說明，加入本研究, 接收抽血, 可能發生抽血部位少量
出血, 紅腫瘀血. 極少情況有暈眩的情形.只需靜坐一段時間, 按壓抽血傷口止血, 局

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部適度冰敷即可. 若仍不適, 可以詢求醫護協助.並由檢體提供者簽署同意書。 
我們將依法, 把任何可辨識身分之記錄, 與個人隱私資料視為機密來處理，不會公
開。研究人員將以一個研究代碼代表受試者身分，此代碼不會顯示其姓名、國民身分證
統一編號、住址等可識別資料。如果發表研究結果，受試者的身分仍將保密。 
其餘參照同意書。 
 
十二、研究人力及相關設備需求。 
1. 收集臨床醫療剩餘檢體腫瘤組織，借助病理部臨床醫事人員。 
工作職責: 由計畫主持人姚明醫師, 以核可IRB 向台大醫院病理部, 申請組織切片. 依
照病理部規範,進行檢體回溯收集. 
2. 其他 收集周邊血液，借助臨床醫事人員  
工作職責: 由計畫主持人姚明醫師或共同主持人羅仕錡醫師解釋研究目的流程後, 由計
畫主持人委託臨床研究護士協助收集受試者同意書.在檢醫部臨床工作人員協助下, 收
集周邊血液, 由研究護士初步處理血液檢體, 並冷凍儲存, 以利計畫主持人姚明醫師或
共同主持人常慧如醫師後續研究所用. 
3. 相關設備需求: 離心機, 4oC 冰箱, 周邊血液單採機. 
 
十三、研究經費需求及其來源。 
經費來源：預計由國衛院經費支持。包括IRB 審查, 病理檢體收集, 周邊血液收及耗材儲
存費用 約NTD 100,000 元 
 
十四、預期成果及主要效益。 
前瞻收集周邊血液, 將抽取單核白血球, 分化為巨噬細胞, 於實驗室中與癌細胞進
行共培養, 並篩選融合細胞, 研究巨噬細胞與癌細胞之交互作用, 及融合細胞之生物特
性 包括纖維化激素表現. 
回溯性收集腫瘤組織切片, 將進行免疫螢光染色, 偵測來自捐髓者的基因表現, 與
腫瘤纖維化程度.  
目前無法預測可能的研究成果。當您的檢體使用於研究時，我們將不會通知您研究
結果或檢體的檢測結果。一般而言，研究所做的檢測分析，大多還不能運用於醫療照護；
但若研究人員認為研究分析的結果，有助於您的醫療照護，也可能會通知您。 
 
十五、研發成果之歸屬及運用。 
由收集的病理及周邊血液臨床檢體, 所產生之研發成果, 歸屬台大醫院與國衛院共同所
有，未來可能將成果發表學術論文等運用 
十六、研究人員利益衝突事項之揭露。 
無利益衝突事項 
 
十七、 參考資料 

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1. 
Yamauchi M, Barker TH, Gibbons DL, Kurie JM. The fibrotic tumor stroma. J Clin Invest 2018; 
128(1): 16-25. 
2. 
Ozdemir BC, Pentcheva-Hoang T, Carstens JL, et al. Depletion of carcinoma-associated 
fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced 
survival. Cancer Cell 2014; 25(6): 719-34. 
3. 
Cassetta L, Pollard JW. Targeting macrophages: therapeutic approaches in cancer. Nat Rev 
Drug Discov 2018; 17(12): 887-904. 
4. 
Poh AR, Ernst M. Targeting Macrophages in Cancer: From Bench to Bedside. Front Oncol 2018; 
8: 49. 
5. 
Kurashige M, Kohara M, Ohshima K, et al. Origin of cancer-associated fibroblasts and 
tumor-associated macrophages in humans after sex-mismatched bone marrow transplantation. 
Commun Biol 2018; 1: 131. 
6. 
Hu H, Jiao F, Han T, Wang LW. Functional significance of macrophages in pancreatic cancer 
biology. Tumour Biol 2015; 36(12): 9119-26. 
7. 
Lu X, Kang Y. Cell fusion as a hidden force in tumor progression. Cancer Res 2009; 69(22): 
8536-9. 
8. 
Gast CE, Silk AD, Zarour L, et al. Cell fusion potentiates tumor heterogeneity and reveals 
circulating hybrid cells that correlate with stage and survival. Sci Adv 2018; 4(9): eaat7828. 
9. 
Johansson CB, Youssef S, Koleckar K, et al. Extensive fusion of haematopoietic cells with 
Purkinje neurons in response to chronic inflammation. Nat Cell Biol 2008; 10(5): 575-83. 
10. Yeh MH, Chang YH, Tsai YC, et al. Bone marrow derived macrophages fuse with intestine 
stromal cells and contribute to chronic fibrosis after radiation. Radiother Oncol 2016; 119(2): 
250-8. 
11. Duelli D, Lazebnik Y. Cell fusion: a hidden enemy? Cancer Cell 2003; 3(5): 445-8. 
12. Lankadasari MB, Mukhopadhyay P, Mohammed S, Harikumar KB. TAMing pancreatic cancer: 
combat with a double edged sword. Mol Cancer 2019; 18(1): 48. 
13. Pawelek JM, Chakraborty AK. Fusion of tumour cells with bone marrow-derived cells: a 
unifying explanation for metastasis. Nat Rev Cancer 2008; 8(5): 377-86. 
14. Chen X, Song E. Turning foes to friends: targeting cancer-associated fibroblasts. Nat Rev 
Drug Discov 2019; 18(2): 99-115. 
15. Murata Y, Kotani T, Ohnishi H, Matozaki T. The CD47-SIRPalpha signalling system: its 
physiological roles and therapeutic application. J Biochem 2014; 155(6): 335-44.