Notch3 signaling promotes colorectal tumor growth by enhancing immunosuppressive cells infiltration in the microenvironment

doi:10.21203/rs.3.rs-1974731/v1

Download PDF

Research Article

Notch3 signaling promotes colorectal tumor growth by enhancing immunosuppressive cells infiltration in the microenvironment

https://doi.org/10.21203/rs.3.rs-1974731/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 16 Jan, 2023

Read the published version in BMC Cancer →

You are reading this latest preprint version

Background: Macrophage infiltration in the tumor microenvironment participates in the regulation of tumor progression. Previous studies have found that Notch signaling pathway is involved in regulating the progression of colorectal cancer (CRC), however, the specific mechanism is still unclear.

Methods: The correlation between Notch signaling pathway and macrophage infiltration was investigated in TCGA database and verified in clinical samples of patients with CRC using immunohistochemistry. Gene Set Enrichment Analysis was used to find out genes related to Notch3 expression. Colony formation assay, and flow cytometry were utilized to test tumor growth and immune cell infiltration in vitro and in vivo.

Results: Using bioinformatics analysis and clinical sample validation, we found that Notch3 was highly expressed in colon tumor tissues compared to adjacent normal tissues, and it participated in regulating the recruitment of macrophages to the tumor microenvironment. Furthermore, we found that the Notch3 expression was positively correlated with the expression of macrophage recruitment-related cytokines in colon tumor tissues. Finally, we demonstrated that depletion of Notch3 had no significant effect on the growth of colon tumor cells in vitro, while, attenuated the growth of colon cancer tumors in vivo. Simultaneous, immunosuppressive cells, macrophages and myeloid-derived suppressor cell (MDSC) infiltration were dramatically reduced in the tumor microenvironment.

Conclusion: Our study illustrated that Notch3 could facilitate the progression of CRC by increasing the infiltration of macrophages and MDSCs to promote the immunosuppressive tumor microenvironment. Targeting Notch3 specifically is a potentially effective treatment for CRC.

Notch3

colorectal tumor

tumor microenvironment

macrophage infiltration

Colorectal cancer (CRC) is one of the most predominant malignant tumors in the digestive system, with mortality and morbidity ranking third and fourth worldwide, respectively [1]. In spite of the rapid development of chemotherapeutic and targeted agents, the prognosis for patients with CRC is still unsatisfactory, with less than 10% of the overall five-year survival rate in the late-stage of CRC [2]. The initiation and progression of CRC is complex, and is driven by multiple genetic and epigenetic alterations involving activation of oncogenes and inactivation of tumor-suppressor genes, which promotes intestinal proliferation, survival, invasion, and chemoresistance [3]. Thus, further investigating the underlying mechanisms of the progression of CRC is a crucial issue to identify novel targets for improving treatment efficacy and prognosis of patients with CRC.

Notch signaling is required for the maintenance of intestinal homeostasis. In mammals, there are four Notch receptors and five Notch ligands [4]. Plenty of research has demonstrated that aberrant activation of Notch signaling plays important role in occurrence and development of various cancers, including CRC. In recent years, increasing evidence has shown that overexpression of Notch signaling is observed in CRC and associated with poor prognosis, and inhibition of Notch signaling might be a potential strategy for the prevention and treatment of CRC [5, 6]. Increased Notch3 expression in human CRC tissues and CRC cell lines has also been reported, and is involved in tumor progression and aggressiveness of CRC [7, 8]. In addition, Notch3 participates in the filtration and activity of immune cells within the tumor microenvironment (TME) [9]. It is now accepted that the tumorigenesis is not only mediated by intrinsic properties in cancer cells but also their interactions with non-cancer cells in TME, such as macrophages, lymphocytes, endothelial cells, and stromal fibroblasts. In particular, tumor-associated macrophages (TAMs) have been illustrated to promote growth and metastasis of colon cancer cells [10]. To date, the underlying mechanism of how Notch3 might interact with TME to promote CRC progression is rarely described.

Herein, we firstly demonstrated that Notch3 participated in regulating the recruitment of macrophages in TME. Bioinformatics analysis indicated Notch3 expression was positively correlated with macrophage recruitment pathways and cytokines in CRC. Moreover, we showed that interference with Notch3 itself had no significant effect on the growth of colon cancer cells in vitro, but significantly inhibited the growth of colon tumors in vivo. Further investigation revealed that interfering Notch3 inhibited the infiltration of macrophages. These findings suggest that Notch3 can promote the development of CRC by enhancing the infiltration of macrophage in the TME, which might shed light on the potential role of Notch3 as a therapeutic target in immunotherapy for CRC.

Cell lines and cell culture

MC38, RAW264.7 and HEK293T cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). All cell lines were authenticated by short tandem repeat genotyping before using. The cell lines were cultured in DMEM (Invitrogen/Thermo Fisher Scientific, MA, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (P/S) (Beyotime Biotechnology, Jiangsu, China) at 37°C with 5% CO₂.

GSEA (Gene Set Enrichment Analysis)

The genes whose expression were positively associated with Notch3 expression in The Cancer Genome Atlas (TCGA)-colorectal carcinoma were picked out using the LinkedOmics browser (http://www.linkedomics.org) [7], which allows the online analysis of TCGA data. Further, we used these genes to do Gene Set Enrichment Analysis. The correlation results were cross-validation using GEPIA [11](Gene Expression Profiling Interactive Analysis) (http://gepia2.cancer-pku.cn/#index).

Flow cytometry analysis

Tumors and blood of tumor-carrying mice were collected. Peripheral blood lymphocytes and tumor-infiltrating lymphocytes were isolated, and red blood cells were lysed using Red Blood Cell Lysis Buffer (Beyotime Biotechnology) for 15 mins according to the manufacturer's instruction manual. Cells were washed and counted using an automated cell counter (Countstar). 1×10⁶ cells were blocked with anti-mouse CD16/32 (BioLegend). To identify the percentage of macrophages and myeloid-derived suppressor cells (MDSCs), cells were stained with antibodies: Ax647-CD11b, FITC-Ly-6G, PE-Gr-1,PerCP/Cy7-F4/80 and PerCP/Cy5.5-Ly-6C, purchased from BioLegend company. Flow cytometry acquisition was performed on CytoFLEX (BD Biosciences), and data were analyzed using CytExpert software (2.4.0.28).

Chemotaxis assay

For analyzing monocyte migration, RAW264.7 cells were seeded (10⁵ cells/ 100 µl DMEM containing 0.1% BSA) onto the top chamber of transwell filters (5-µm; Corning). Filters were placed in a 24-well plate that contains condition medium isolated from the vehicle- or Notch3-depleted MC38 cell lines. 24 hours following incubation, cells in the lower chamber were isolated and counted. Samples were run in triplicates for each group.

Histology and Immunohistochemistry (IHC)

Human CRC tissue samples were obtained from The First Affiliated Hospital of Anhui Medical University. All samples were obtained with informed consent, and the study was approved by the Ethics Committee of Anhui Medical University, as well as the principles expressed in the Declaration of Helsinki. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded human tissue sections using a biotin-avidin method as described before[12]. Sections were developed with DAB and counterstained with hematoxylin. Whole-slide imaging was taken using PANNORAMIC 1000 (3D-HISTECH, Hungary). Quantification of NOTCH3 IHC chromogen intensity was performed as previously described [13]. Briefly, staining intensity (0, 1, 2, 3) and percentage of positive cells among cancer (0–25% recorded as 1, 25–50% as 2, 50–75% as 3 and > 75% as 4) were evaluated by a pathologist. The final immunoreactive scores were calculated by multiplying the two numbers as previously described [13].

TCGA and GEO data analysis

Clinical data and RNA expression data with normalized RSEM values of the colorectal carcinoma datasets (TCGA-COAD) were retrieved from FireBrowse (http://firebrowse.org). The patients with CRC had associated comprehensive follow-up records, RNA expression data and records of clinical treatment.

Survival curve

The overall survival of the four Notch receptors was assessed by the TISIDB, a web portal for tumor and immune system interaction, which integrates multiple heterogeneous data types[14].

Tumor-infiltrating immune cell analysis

TIMER2.0 (http://timer.cistrome.org/), an online tool, which is a comprehensive resource for systematical analysis of immune infiltrates across diverse cancer types, was used to analyze tumor-infiltrating macrophage cells in TCGA-COAD patients [15].

Western blot

Western blot was performed as previously described[16]. Briefly, cells were lysed on ice for 30 mins using cell lysis buffer (50mM Tris HCl, pH 7.4, with 250mM NaCl, 1 mM EDTA, 50mM NaF, and 0.5% Triton X-100, together with protease inhibitors). Then the lysis was centrifugation at 12000 rpm for 15 mins at 4℃. The protein concentration of each sample was determined using Bradford Kit (Sangon Biotech. Shanghai, China). For immunoblotting, proteins were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). Immunoblots were developed in Western Lightning Chemiluminescence Reagent Plus (Advansta, Menlo Park, CA, USA).

Colony formation assay

For colony formation assays, approximately 20000 cells per well were seeded into 12-well plates and treated with various concentrations of drugs for 72 hrs. Cells were then fixed in 4% formaldehyde for 30 mins and stained with 0.1% (w/v) crystal violet solution.

Lentivirus production and infection

Lentiviral particles were packaged using HEK293T cells. Briefly, 5×10⁵ cells were seed in a 60 mm dish in complete DMEM culture medium one day before transfection. On the day of transfection, 18 µl PEI, 3 µg of lentiviral construct as well as 1.5 µg of psPAX2 and pMD2.G were pipet directly into 182 µl serum and antibiotics-free DMEM medium. Then vortexed and added to HEK293T cells in a drop-wise manner 15 mins later. The virus was collected 48 hours later and filtered using non-pyrogenic filters (0.45 mm, Merck Millipore, Billerica, MA, USA). Diluted lentivirus and 8 µg /ml polybrene (Sigma-Aldrich, St.Louis, MO, USA) were added to targeting cells. For stable cell selection, puromycin (1 µg/ml) was used.

Statistical analysis

Data analyses were performed with GraphPad Prism. P value was determined using two-way ANOVA for tumor growth, Log-rank test for survival, and two-tailed t-tests for other analyses. p < 0.05 was considered as statistically significant.

Notch3 expression was significantly positively correlated with macrophage infiltration

In order to explore the relationship between Notch signaling pathway and macrophage infiltration in CRC, TCGA databases were used (Fig. 1A). Firstly, it has been shown that Notch receptors expression was positively correlated with the macrophage infiltration in CRC (Fig. 1A). Moreover, similar results were obtained using different calculation methods of immune cell infiltration (Fig. 1A), suggesting the expression of Notch receptors is closely related to the infiltration of macrophages in CRC. By comparing the positive correlation efficient between Notch receptors expression and macrophage infiltration, we found Notch2 and Notch3 were more efficient (Fig. 1B, Fig. S1A), indicating these two Notch receptors are more likely to be involved in regulating macrophage infiltration. Then, the expression levels of Notch receptors in colon cancer tumors and normal tissues were analyzed, and we showed that only the level of Notch3 was highly expressed in tumor tissues (Fig. 1C). At the same time, the high expression of Notch3 significantly positively correlated with the poor prognosis of patients with CRC (Fig. 1D, Fig. S1B). In summary, we found that Notch3 receptor was positively correlated with macrophage infiltration in CRC as well as the poor prognosis of patients.

Notch3 expression was positively correlated with the degree of macrophage infiltration in colorectal carcinoma tissue.

To confirm if Notch3 protein expression was positively correlated with macrophage infiltration, paraffin colorectal carcinoma specimens were collected and stained with Notch3 and CD68, a commonly used macrophage marker. The expression levels of Notch3 were gradually increased as the tumor progressed (Fig. 2A). In addition, Notch3 was highly expressed in colon cancer tissues compared to normal tissues (Fig. 2B, Fig. S2A). Next, we stained the serial sections for Notch3 and CD68, respectively, and found the number of CD68-positive cells was significantly increased in the areas with high Notch3 expression (Fig. 2C, Fig. S2B). Statistically, a significantly positive correlation was observed between Notch3 expression and macrophage infiltration (Fig. 2D). Taken together, our results indicated that Notch3 protein expression was significantly positively correlated with the degree of macrophage infiltration in colon cancer tumor tissue.

Notch3 was positive correlated with the expression of macrophage recruitment-related cytokines.

Next, to further investigate how Notch3 was involved in regulating macrophage infiltration, TCGA database was utilized. Genes that were significantly positively associated with Notch3 expression in colorectal carcinoma were picked out and GSEA enrichment were performed. The results showed that genes related to chemokines and cytokine-receptor binding pathways were significantly enriched (Fig. 3A and B), implying that Notch3 plays an essential role in the macrophage recruitment by regulating the cytokines expression. Then, we analyzed the current known relationship between Notch3 and the expression of macrophage recruitment-related cytokines and found the expression levels of cytokines including CSF1, CXCL12, CCL2 were significantly positively correlated with the expression levels of Notch3, especially CSF1, which is a well-known cytokine involved in promoting macrophage recruitment (Fig. 3C, Fig. S3A). To further verify this result, another database- GEPIA was exploited to analyze the above-mentioned cytokines and consistent results were obtained (Fig. S3B). Taken together, we demonstrated that Notch3 was significantly positively correlated with the expression of macrophage recruitment-related factors, suggesting that Notch3 can regulate the infiltration of macrophage in tumor tissues by regulating the expression of the above-mentioned cytokines, such as CSF1.

Interference with Notch3 attenuated the colon tumor growth and decreased macrophage infiltration in vivo.

In order to test whether Notch3 was involved in regulating the recruitment of macrophages in vivo, an MC38 cell line with stable knockdown of Notch3 was constructed (Fig. 4A). The colony formation assay showed that interference with Notch3 expression in vitro had little effect on cell proliferation (Fig. 4B). Next, we injected control and Notch3 stable knockdown MC38 cells on both sides of C57BL/6 mice. Surprisingly, interference with Notch3 significantly inhibited the growth of transplanted tumors (Fig. 4C-E). The above results indicated that Notch3 mainly promoted the development of CRC by regulating the tumor microenvironment. Furthermore, a flow cytometry experiment was conducted to detect macrophage in the tumor microenvironment of transplanted tumors. The results showed that interference with Notch3 significantly reduced the proportion of macrophage in tumor tissues (Fig. 4F, G, Fig. S4A). Meanwhile, the proportion of macrophage was analyzed in the peripheral blood of mice, and no significant changes were observed (Fig. S4B). Altogether, our results suggested that Notch3 participated in regulating the recruitment of macrophage in colon cancer, thereby regulating tumor progression.

In the present work, we have shown that the expressions of Notch receptors were positively correlated with the in infiltration of macrophage, among which only Notch3 was highly expressed in colorectal tumor tissues and associated with poor prognosis of patients with CRC. These findings were also confirmed in clinical samples in which Notch3 expression was positively related to the extent of macrophage infiltration. Bioinformatics analysis revealed that Notch3 was found to regulate certain pathways and cytokines involving macrophage recruitment. A notable finding was that inhibition of Notch3 had no influences on colorectal cancer cell proliferation in vitro, whereas interfering with Notch3 significantly decreased tumor growth in vivo. Mechanically, interference with Notch3 significantly reduced the proportion of macrophage in tumor tissue while no obvious changes were detected in peripheral blood of mice, suggesting Notch3 might participate in the regulation of macrophage recruitment and thus promote tumor growth of CRC.

Notch3 is reported to be overexpressed in human CRC and CRC cell lines [17]. Despite the role of Notch3 in CRC has been relatively less investigated, increasing focus is paid on the essential contribution of Notch3 to CRC progression. Ozawa et al. showed significant correlation between nuclear Notch3 expression and tumor recurrence in stage II and stage III CRC [18]. Recently, Varga et al. have indicated that Notch3 expression in CRC was upregulated with tumor stage and correlated to tumor invasion and worse overall survival [19]. In addition, several studies demonstrated that forced Notch3 expression accelerated tumor formation in CRC xenografts while inactivation of Notch3 expression decreased cell proliferation, the clonogenic potential of CRC cells and tumor growth [7, 20, 21]. In consistence with these findings, our study demonstrated that Notch3 plays a crucial role in promoting the growth of CRC. To be noted, we showed that inhibition of Notch3 had no obvious influences on colorectal cancer cell proliferation in vitro. The discrepancy could be attributed to cell-type or context-specific. However, the antitumor effect in vivo has shed lights on the indispensable role of TME which might interact with Notch3 in the development of CRC.

Accumulating evidence has shown the dynamic composition of TME participates in tumor initiation, progression and drug resistance. TAMs as a major component of immune cells in TME play complex roles in tumor development, which can promote or inhibit tumor invasion mediating via interacting with other immune cells and cancer cells. Macrophage infiltration are generally associated with poor clinical outcomes in many solid tumors, including CRC [22]. Using a pre-clinical immunocompetent orthotopic CRC mouse model, Trimaglio et al. revealed a significant role of colon microenvironment in the regulation of CRC immune responses [23]. Recently, a study by Algars et al. focusing on the prognostic role of TAMs in CRC showed that different patterns of macrophage and lymphatic vessels can predict the prognosis according to different stages of CRC [24]. Yin et al. reported that high numbers of TAMs in CRC tissues correlated to chemoresistance by regulating the IL6R/STAT3/miR-204-5p axis, and was associated with poor prognosis in patients with CRC [25].

Notch signaling clearly plays important roles in TAMs, either to promote or suppress tumor growth. The dysregulation of Notch signaling can mediate the interactions between cancer cells and their surrounding TME [26]. Besides, Notch signaling participates in the regulation of macrophage activation and inflammatory processes. Ye et al. demonstrated that Notch and Wnt signaling regulated the differentiation of TAMs in hepatocellular carcinoma. progression and hepatic metastasis of CRC [27]. Notch signaling has also been described to positively regulate cytokines produced by macrophages which promotes cancer growth and metastasis [28]. Particularly, Notch3 is selectively upregulated during macrophage differentiation and contributes to the pro-inflammatory activation of macrophages [9]. Li et al. recently reported that Notch3 participated in the infiltration and activity of immune cells in TME which contributes to poor prognosis of gastric cancer [29]. Here, we have demonstrated that the expression of Notch3 was positively associated with the level of soluble tumor-derived chemotactic factors, such as CSF1, CXCL12 and CCL2, which are chemotactic stimulus for macrophage recruitment. macrophage can be recruited to the tumor periphery by CSF-1 produced by CRC cells, and further secret factors to promote cell invasion [30]. CSF1 is required for accumulation of TAM and plays a critical role in macrophage differentiation[31]. In Diffuse large B-cell lymphoma, Huang et al. showed that Notch signaling can regulate the expressions of CSF1 and CCL2, which contributes to TAM modulation and progression [32]. CCL2 as a member of the C-C chemokine family is reported to correlate with the infiltration of TAMs and poor prognosis in many tumors [33]. Chun et al. found the level of CCL2 increased in patients with CRC and neutralization of CCL2 halted tumor progression in a mouse model of inflammation-associated CRC [34]. Shen et al. utilized a breast cancer model and demonstrated that Notch can regulate cancer cell expression of IL1β and CCL2 in vivo experiment, and in vivo models further confirmed an association between Notch-dependent cytokine production and TAM recruitment [35]. It is known that CXCL12 upregulation promotes the recruitment of Tregs and the infiltration of macrophage. Accumulating evidence has shown CXCL12-CXCR4 axis plays a critical role in CRC progression and metastasis [36, 37]. Besides, Zboralski et al. illustrated a key role of CXCL12-CXCR4 axis in conferring resistance to checkpoint inhibitors and blockage of CXCL12 enhanced the efficacy of anti-PD-L1 immunotherapy in a murine model of CRC [38].

In conclusion, our study elucidated an important role of Notch3 as a regulator in macrophage recruitment and microenvironment in CRC. Notch3 can promote CRC progression via upregulation of macrophage infiltration in TME. Thus, Notch3 might be a promising target in combination with immunotherapy for CRC treatment.

CRC

Colorectal cancer

IHC

immunohistochemistry

TAMs

tumor-associated macrophages

TCGA

The Cancer Genome Atlas

TME

tumor microenvironment

Ethics approval and consent to participate

The study was approved by the Ethics Committee of Anhui Medical University. All participation gave written informed consent in accordance with the Declaration of Helsinki. All experimental methods were conducted in accordance with the Animal Care and Use Regulations of China. All methods were reported in accordance with ARRIVE guidelines.

We would like to thank the TCGA database, GEPIA database, and TIMER for providing their platforms and for their contributions to constructing and uploading their valuable datasets.

Consent for publication

Not applicable.

Availability of data and materials

The public datasets could be downloaded at http://www.linkedomics.org (cohort: TCGA) and http://gepia2.cancer-pku.cn/#index (cohort: GEPIA). Other data analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

None

Funding

This study was supported by the Fundamental Research Funds for the Central Universities (grant number WK9110000131 to XY)

Authors' contributions

WW, YL and YX conceived and designed this project; KH performed the experiments and wrote the original draft; WL, JF, YC and LG collected and analyzed the data; All authors contributed to the article and approved the final version of the manuscript.

Acknowledgements

None

Rawla, P., T. Sunkara, and A. Barsouk, Epidemiology of colorectal cancer: incidence, mortality, survival, and risk factors. Przeglad gastroenterologiczny, 2019. 14(2): p. 89–103.
Siegel, R.L., K.D. Miller, S.A. Fedewa, D.J. Ahnen, R.G.S. Meester, A. Barzi, and A. Jemal, Colorectal cancer statistics, 2017. CA Cancer J Clin, 2017. 67(3): p. 177–193.
Müller, M.F., A.E. Ibrahim, and M.J. Arends, Molecular pathological classification of colorectal cancer. Virchows Arch, 2016. 469(2): p. 125–34.
Demitrack, E.S. and L.C. Samuelson, Notch regulation of gastrointestinal stem cells. J Physiol, 2016. 594(17): p. 4791–803.
Sonoshita, M., M. Aoki, H. Fuwa, K. Aoki, H. Hosogi, Y. Sakai, H. Hashida, A. Takabayashi, M. Sasaki, S. Robine, K. Itoh, K. Yoshioka, F. Kakizaki, T. Kitamura, M. Oshima, and M.M. Taketo, Suppression of colon cancer metastasis by Aes through inhibition of Notch signaling. Cancer Cell, 2011. 19(1): p. 125–37.
Suman, S., T.P. Das, M.K. Ankem, and C. Damodaran, Targeting Notch Signaling in Colorectal Cancer. Curr Colorectal Cancer Rep, 2014. 10(4): p. 411–416.
Serafin, V., L. Persano, L. Moserle, G. Esposito, M. Ghisi, M. Curtarello, L. Bonanno, M. Masiero, D. Ribatti, M. Stürzl, E. Naschberger, R.S. Croner, A.M. Jubb, A.L. Harris, H. Koeppen, A. Amadori, and S. Indraccolo, Notch3 signalling promotes tumour growth in colorectal cancer. J Pathol, 2011. 224(4): p. 448–60.
Liu, K., J.-F. Wang, Y. Zhan, D.-L. Kong, and C. Wang, Prognosis model of colorectal cancer patients based on NOTCH3, KMT2C, and CREBBP mutations. Journal of Gastrointestinal Oncology, 2021. 12(1): p. 79–88.
López-López, S., E.M. Monsalve, M.J. Romero de Ávila, J. González-Gómez, N. Hernández de León, F. Ruiz-Marcos, V. Baladrón, M.L. Nueda, M.J. García-León, I. Screpanti, M.P. Felli, J. Laborda, J.J. García-Ramírez, and M.J.M. Díaz-Guerra, NOTCH3 signaling is essential for NF-κB activation in TLR-activated macrophages. Sci Rep, 2020. 10(1): p. 14839.
Wang, H., T. Tian, and J. Zhang, Tumor-Associated Macrophages (TAMs) in Colorectal Cancer (CRC): From Mechanism to Therapy and Prognosis. International journal of molecular sciences, 2021. 22(16): p. 8470.
Li, C., Z. Tang, W. Zhang, Z. Ye, and F. Liu, GEPIA2021: integrating multiple deconvolution-based analysis into GEPIA. Nucleic Acids Res, 2021. 49(W1): p. W242-W246.
Yuan, X., L. Huang, W. Luo, Y. Zhao, B. Nashan, F. Yu, and Y. Liu, Diagnostic and Prognostic Significances of SOX9 in Thymic Epithelial Tumor. Front Oncol, 2021. 11: p. 708735.
Li, Z., P. Razavi, Q. Li, W. Toy, B. Liu, C. Ping, W. Hsieh, F. Sanchez-Vega, D.N. Brown, A.F. Da Cruz Paula, L. Morris, P. Selenica, E. Eichenberger, R. Shen, N. Schultz, N. Rosen, M. Scaltriti, E. Brogi, J. Baselga, J.S. Reis-Filho, et al., Loss of the FAT1 Tumor Suppressor Promotes Resistance to CDK4/6 Inhibitors via the Hippo Pathway. Cancer Cell, 2018. 34(6): p. 893–905 e8.
Ru, B., C.N. Wong, Y. Tong, J.Y. Zhong, S.S.W. Zhong, W.C. Wu, K.C. Chu, C.Y. Wong, C.Y. Lau, I. Chen, N.W. Chan, and J. Zhang, TISIDB: an integrated repository portal for tumor-immune system interactions. Bioinformatics, 2019. 35(20): p. 4200–4202.
Li, T., J. Fu, Z. Zeng, D. Cohen, J. Li, Q. Chen, B. Li, and X.S. Liu, TIMER2.0 for analysis of tumor-infiltrating immune cells. Nucleic Acids Res, 2020. 48(W1): p. W509-W514.
Huang, K., S. Chen, R. Xie, P. Jiang, C. Yu, J. Fang, X. Liu, and F. Yu, Identification of three predictors of gastric cancer progression and prognosis. FEBS Open Bio, 2020. 10(9): p. 1891–1899.
Aburjania, Z., S. Jang, J. Whitt, R. Jaskula-Stzul, H. Chen, and J.B. Rose, The Role of Notch3 in Cancer. The oncologist, 2018. 23(8): p. 900–911.
Ozawa, T., S. Kazama, T. Akiyoshi, K. Murono, S. Yoneyama, T. Tanaka, J. Tanaka, T. Kiyomatsu, K. Kawai, H. Nozawa, T. Kanazawa, H. Yamaguchi, S. Ishihara, E. Sunami, J. Kitayama, T. Morikawa, M. Fukayama, and T. Watanabe, Nuclear Notch3 expression is associated with tumor recurrence in patients with stage II and III colorectal cancer. Ann Surg Oncol, 2014. 21(8): p. 2650-8.
Varga, J., A. Nicolas, V. Petrocelli, M. Pesic, A. Mahmoud, B.E. Michels, E. Etlioglu, D. Yepes, B. Häupl, P.K. Ziegler, K. Bankov, P.J. Wild, S. Wanninger, H. Medyouf, H.F. Farin, S. Tejpar, T. Oellerich, J. Ruland, C.W. Siebel, and F.R. Greten, AKT-dependent NOTCH3 activation drives tumor progression in a model of mesenchymal colorectal cancer. J Exp Med, 2020. 217(10).
Pastò, A., V. Serafin, G. Pilotto, C. Lago, C. Bellio, L. Trusolino, A. Bertotti, T. Hoey, M. Plateroti, G. Esposito, M. Pinazza, M. Agostini, D. Nitti, A. Amadori, and S. Indraccolo, NOTCH3 signaling regulates MUSASHI-1 expression in metastatic colorectal cancer cells. Cancer Res, 2014. 74(7): p. 2106–18.
Koch, U. and F. Radtke, A third Notch in colorectal cancer progression and metastasis. J Exp Med, 2020. 217(10).
Wang, H., T. Tian, and J. Zhang, Tumor-Associated Macrophages (TAMs) in Colorectal Cancer (CRC): From Mechanism to Therapy and Prognosis. Int J Mol Sci, 2021. 22(16).
Trimaglio, G., A.F. Tilkin-Mariamé, V. Feliu, F. Lauzéral-Vizcaino, M. Tosolini, C. Valle, M. Ayyoub, O. Neyrolles, N. Vergnolle, Y. Rombouts, and C. Devaud, Colon-specific immune microenvironment regulates cancer progression versus rejection. Oncoimmunology, 2020. 9(1): p. 1790125.
Ålgars, A., L. Kemppinen, R. Fair-Mäkelä, H. Mustonen, C. Haglund, and S. Jalkanen, Stage I-IV Colorectal Cancer Prognosis Can Be Predicted by Type and Number of Intratumoral Macrophages and CLEVER-1(+) Vessel Density. Cancers (Basel), 2021. 13(23).
Yin, Y., S. Yao, Y. Hu, Y. Feng, M. Li, Z. Bian, J. Zhang, Y. Qin, X. Qi, L. Zhou, B. Fei, J. Zou, D. Hua, and Z. Huang, The Immune-microenvironment Confers Chemoresistance of Colorectal Cancer through Macrophage-Derived IL6. Clin Cancer Res, 2017. 23(23): p. 7375–7387.
Colombo, M., L. Mirandola, M. Chiriva-Internati, A. Basile, M. Locati, E. Lesma, R. Chiaramonte, and N. Platonova, Cancer Cells Exploit Notch Signaling to Redefine a Supportive Cytokine Milieu. Front Immunol, 2018. 9: p. 1823.
Ye, Y.C., J.L. Zhao, Y.T. Lu, C.C. Gao, Y. Yang, S.Q. Liang, Y.Y. Lu, L. Wang, S.Q. Yue, K.F. Dou, H.Y. Qin, and H. Han, NOTCH Signaling via WNT Regulates the Proliferation of Alternative, CCR2-Independent Tumor-Associated Macrophages in Hepatocellular Carcinoma. Cancer Res, 2019. 79(16): p. 4160–4172.
Hossain, F., S. Majumder, D.A. Ucar, P.C. Rodriguez, T.E. Golde, L.M. Minter, B.A. Osborne, and L. Miele, Notch Signaling in Myeloid Cells as a Regulator of Tumor Immune Responses. Front Immunol, 2018. 9: p. 1288.
Cui, Y., Q. Li, W. Li, Y. Wang, F. Lv, X. Shi, Z. Tang, Z. Shen, Y. Hou, H. Zhang, B. Mao, and T. Liu, NOTCH3 is a Prognostic Factor and Is Correlated With Immune Tolerance in Gastric Cancer. Front Oncol, 2020. 10: p. 574937.
Gallo, G., G. Vescio, G. De Paola, and G. Sammarco, Therapeutic Targets and Tumor Microenvironment in Colorectal Cancer. J Clin Med, 2021. 10(11).
Lin, W., D. Xu, C.D. Austin, P. Caplazi, K. Senger, Y. Sun, S. Jeet, J. Young, D. Delarosa, E. Suto, Z. Huang, J. Zhang, D. Yan, C. Corzo, K. Barck, S. Rajan, C. Looney, V. Gandham, J. Lesch, W.C. Liang, et al., Function of CSF1 and IL34 in Macrophage Homeostasis, Inflammation, and Cancer. Front Immunol, 2019. 10: p. 2019.
Huang, Y.H., K. Cai, P.P. Xu, L. Wang, C.X. Huang, Y. Fang, S. Cheng, X.J. Sun, F. Liu, J.Y. Huang, M.M. Ji, and W.L. Zhao, CREBBP/EP300 mutations promoted tumor progression in diffuse large B-cell lymphoma through altering tumor-associated macrophage polarization via FBXW7-NOTCH-CCL2/CSF1 axis. Signal Transduct Target Ther, 2021. 6(1): p. 10.
Pathria, P., T.L. Louis, and J.A. Varner, Targeting Tumor-Associated Macrophages in Cancer. Trends Immunol, 2019. 40(4): p. 310–327.
Chun, E., S. Lavoie, M. Michaud, C.A. Gallini, J. Kim, G. Soucy, R. Odze, J.N. Glickman, and W.S. Garrett, CCL2 Promotes Colorectal Carcinogenesis by Enhancing Polymorphonuclear Myeloid-Derived Suppressor Cell Population and Function. Cell Rep, 2015. 12(2): p. 244–57.
Shen, Q., B. Cohen, W. Zheng, R. Rahbar, B. Martin, K. Murakami, S. Lamorte, P. Thompson, H. Berman, J.C. Zuniga-Pflucker, P.S. Ohashi, and M. Reedijk, Notch Shapes the Innate Immunophenotype in Breast Cancer. Cancer Discov, 2017. 7(11): p. 1320–1335.
Khare, T., M. Bissonnette, and S. Khare, CXCL12-CXCR4/CXCR7 Axis in Colorectal Cancer: Therapeutic Target in Preclinical and Clinical Studies. Int J Mol Sci, 2021. 22(14).
Yu, X., D. Wang, X. Wang, S. Sun, Y. Zhang, S. Wang, R. Miao, X. Xu, and X. Qu, CXCL12/CXCR4 promotes inflammation-driven colorectal cancer progression through activation of RhoA signaling by sponging miR-133a-3p. J Exp Clin Cancer Res, 2019. 38(1): p. 32.
Zboralski, D., K. Hoehlig, D. Eulberg, A. Frömming, and A. Vater, Increasing Tumor-Infiltrating T Cells through Inhibition of CXCL12 with NOX-A12 Synergizes with PD-1 Blockade. Cancer Immunol Res, 2017. 5(11): p. 950–956.

No competing interests reported.

FigS1.jpg
Figure S1. Notch3 expression was significantly positively correlated with macrophages infiltration. A. Representative correlation results analyzed using CIBERSORT-ABS. B. Survival curves of patients with colorectal carcinoma from the TCGA database stratified according to expression levels of Notch1,2,4.
FigS2.jpg
Figure S2. Notch3 expression was positively correlated with the degree of macrophage infiltration in colorectal carcinoma tissue. A. IHC score standard of Notch3 in colon cancer. C. Notch3 and CD68 staining of the colon cancer tissue continuous section from patient #2.
FigS3.jpg
Figure S3. Notch3 expression was positively correlated with the macrophage recruitment-related cytokines expression. A. The relation between Notch3 and macrophage recruitment-related genes analyzed using LinkedOmics database (http://www.linkedomics.org/login.php). B. The relationship between Notch3 and macrophage recruitment-related genes analyzed using GEPIA2 database (http://gepia2.cancer-pku.cn/#index).
FigS4.jpg
Figure S4. Interference Notch3 attenuated the colon tumor growth and decreased macrophage infiltration in vivo. A. Gating strategy for flow cytometry. B. Flow cytometry analysis of blood from MC38 tumors carrying mice. C. The quantification of the results in B.

Download PDF

Journal Publication

published 16 Jan, 2023

Read the published version in BMC Cancer →

Editorial decision: Major revision
18 Oct, 2022
Reviews received at journal
17 Oct, 2022
Reviews received at journal
10 Oct, 2022
Reviewers agreed at journal
28 Sep, 2022
Reviewers agreed at journal
21 Sep, 2022
Reviewers invited by journal
01 Sep, 2022
Editor assigned by journal
01 Sep, 2022
Editor invited by journal
31 Aug, 2022
Submission checks completed at journal
31 Aug, 2022
First submitted to journal
18 Aug, 2022

You are reading this latest preprint version

Notch3 signaling promotes colorectal tumor growth by enhancing immunosuppressive cells infiltration in the microenvironment

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Methods

Cell lines and cell culture

GSEA (Gene Set Enrichment Analysis)

Flow cytometry analysis

Chemotaxis assay

Histology and Immunohistochemistry (IHC)

TCGA and GEO data analysis

Survival curve

Tumor-infiltrating immune cell analysis

Western blot

Colony formation assay

Lentivirus production and infection

Statistical analysis

Results

Notch3 expression was significantly positively correlated with macrophage infiltration

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1