Expansion of cancer stem cell pool initiates lung cancer recurrence before angiogenesis Lei Lia,b,1, Jiang-Chao Lic,1, Hong Yangd,e,1, Xu Zhange, Lu-Lu Liuf, Yan Lig, Ting-Ting Zenga,b, Ying-Hui Zhua,b, Xiao-Dong Lie, Yan Lia,b, Dan Xiea,b,LiFuh,i, and Xin-Yuan Guana,b,j,2 aState Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, 510060 Guangzhou, China; bCollaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, 510060 Guangzhou, China; cVascular Biology Research Institute, School of Basic Courses, Guangdong Pharmaceutical University, 510006 Guangzhou, China; dGuangdong Esophageal Cancer Research Institute, Sun Yat-sen University Cancer Center, 510060 Guangzhou, China; eDepartment of Thoracic Oncology, Sun Yat-sen University Cancer Center, 510060 Guangzhou, China; fCancer Biotherapy Center, The First Affiliated Hospital of Zhejiang University, 310003 Hangzhou, China; gGuangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research, Shenzhen Key Laboratory of Cell Microenvironment, Department of Biology, Southern University of Science and Technology, 518055 Shenzhen, China; hDepartment of Pharmacology, Shenzhen University School of Medicine, 518060 Shenzhen, China; iCarson International Cancer Center, Shenzhen University School of Medicine, 518060 Shenzhen, China; and jDepartment of Clinical Oncology, The University of Hong Kong, 852 Hong Kong, China Edited by Dennis A. Carson, University of California, San Diego, La Jolla, CA, and approved August 7, 2018 (received for review April 12, 2018) Angiogenesis is essential in the early stage of solid tumor recur- Therefore, research progress on the mechanisms about tumor mi- rence, but how a suspensive tumor is reactivated before angiogen- croenvironment regulating relapse will provide promising targets for esis is mostly unknown. Herein, we stumble across an interesting the treatment of recurrence and metastasis. phenomenon that s.c. xenografting human lung cancer tissues can Cancer stem cells (CSCs) are generally considered to be re- awaken the s.c. suspensive tumor in nude mice. We further found sponsible for tumorigenesis and cancer metastasis (12, 13). Im- that a high level of insulin-like growth factor 1 (IGF1) was mainly portantly, the existence of CSCs may account for the relapse of responsible for triggering the transition from suspensive tumor cancer, especially after radiotherapy and chemotherapy. However, to progressive tumor in this model. The s.c. suspensive tumor is it is still debatable if CSCs differ from indolent tumor cells. CSCs characterized with growth arrest, avascularity, and a steady-state + are deemed as latent cells due to their low proliferation rate, and level of proliferating and apoptotic cells. Intriguingly, CD133 lung resistance to chemotherapy and radiotherapy, which is similar to cancer stem cells (LCSCs) are highly enriched in suspensive tumor the characteristics of dormant cells. However, it could be argued MEDICAL SCIENCES compared with progressive tumor. Mechanistically, high IGF1 ini- that although dormant cells can repopulate a whole system, they tiates LCSCs self-renewal from asymmetry to symmetry via the ac- will not be considered as stem cells unless they possess self- β tivation of a PI3K/Akt/ -catenin axis. Next, the expansion of LCSC renewal ability (14). CSCs have a unique biological process for pool promotes angiogenesis by increasing the production of CXCL1 + self-renewal that one CSC may produce one daughter CSC via and PlGF in CD133 LCSCs, which results in lung cancer recurrence. asymmetric cell division or two daughter CSCs via symmetric cell Clinically, a high level of serum IGF1 in lung cancer patients after division, which ensures the CSC population to be maintained or orthotopic lung cancer resection as an unfavorable factor is strongly expanded for long-term clonal growth (15). However, whether the correlated with the high rate of recurrence and indicates an adverse progression-free survival. Vice versa, blocking IGF1 or CXCL1/PlGF imbalance of self-renewal in CSCs is responsible for cancer relapse with neutralizing antibodies can prevent the reactivation of a sus- has not been investigated. pensive tumor induced by IGF1 stimulation in the mouse model. In this study, we demonstrated that lung cancer stem cells Collectively, the expansion of LCSC pool before angiogenesis in- (LCSCs) maintained a dynamic equilibrium of tumor cell duced by IGF1 is a key checkpoint during the initiation of cancer relapse, and targeting serum IGF1 may be a promising treatment for Significance preventing recurrence in lung cancer patients. Latent tumor cells are the crucial reason of tumor recurrence and angiogenesis | recurrence | IGF-1 | cancer stem cell | self-renewal the death of cancer patients. Preventing latent tumor relapse can prolong patients’ survival and have a long time surviving ancer recurrence, a major cause of cancer death, can be preceded with latent tumor cells. Here, we describe a lung cancer sus- Cby an interlude, termed tumor dormancy, that can last years or pensive tumor model in mouse and find that a high level of even decades without clinical symptoms (1). The incidence of cancer cancer stem cells undergoing asymmetric cell division in latent recurrence after aggressive cancer surgery is also high (2). Recent tumor is the key issue to reactivate a suspensive tumor. The studies have indicated that minimal indolent tumors were the origins results clearly delineate the state of latent tumor in vivo. A high of metastasis and recurrence (3–5). However, the molecular mecha- level of serum IGF-1 can induce the suspensive-to-progressive tumor transition though promoting CSCs symmetric division, nisms underlying tumor dormancy and rebirth are far from clear. which illuminate a key checkpoint of cancer relapse before an- Currently, there are two distinct theories to explain tumor dor- giogenesis, highlighting a potential therapeutic target for pre- mancy: Cellular dormancy describes that cells enter a quiescence venting tumor recurrence. statuswheregrowthisarrestedinG0/G1 phase of cell cycle, and cells are completely inactive and asymptomatic (6); population Author contributions: D.X., L.F., and X.-Y.G. designed research; L.L., J.-C.L., H.Y., L.-L.L., dormancy presents that a cluster of tumor cells maintain a balance T.-T.Z., Y.-H.Z., and Yan Li performed research; X.Z., Yan Li, and X.-D.L. contributed new between proliferation and apoptosis without expansion (7). These reagents/analytic tools; L.L., J.-C.L., and H.Y. analyzed data; and L.L. wrote the paper. dormant switches are two hurdles that must be overcome for tumor The authors declare no conflict of interest. recurrence and metastasis initiation. When tumor cells disseminate This article is a PNAS Direct Submission. into a new site, the interaction between the tumor cells and their Published under the PNAS license. microenvironment determines whether the cells enter proliferation 1L.L., J.-C.L., and H.Y. contributed equally to this work. or dormancy (8). Arrested cells can come back as recurrent tumor 2To whom correspondence should be addressed. Email: [email protected]. when the microenvironment changes after a long-time latency. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Angiogenesis, immune response, and cytokine network may account 1073/pnas.1806219115/-/DCSupplemental. for the transition between indolent and aggressive tumor (9–11). www.pnas.org/cgi/doi/10.1073/pnas.1806219115 PNAS Latest Articles | 1of10 Downloaded by guest on September 29, 2021 proliferation and death via asymmetric cell division in indolent such as promoting cell proliferation, stimulating angiogenesis, and tumor. However, the division of LCSCs could be switched to regulating cell stemness, and administrated through tail i.v. in- symmetric cell division by a high level of insulin-like growth factor jection with a high dose for per factor (Fig. 1G and SI Appendix, 1 (IGF1), which led to the rapid expansion of LCSC population Table S2). Results showed that only group 1 factors, including and angiogenesis, resulting in lung cancer recurrence. IGF1, LIF, and SCF, could effectively induce the transition from suspensive tumor to progressive tumor (Fig. 1H). Separated tests Results further showed that IGF1 (2 μg per mouse) was only responsible Xenografting Human Lung Cancer Tissues Induces the Tumor Recurrence for the initiation of the tumor relapse (Fig. 1I). In addition, the in Mouse. Patient-derived xenograft (PDX) maintains the histo- human IGF1 levels in mice serum were also compared by ELISA pathological and molecular characteristics of the parental tumor, between mice with and without the human NSCLC PDX-triggered offering an exciting tool for studying targeted therapies (16, 17). recurrence. Results showed that the average serum IGF1 levels of Interestingly, we happened upon an interesting phenomenon that mice with recurrent tumors were significantly higher than that in s.c. xenografting certain human lung cancer tissues in nude mice mice with suspensive tumors (Fig. 1J). could induce the recurrence of suspensive xenograft tumor derived Moreover, we also tested the responses of suspensive tumors to from nonsmall-cell lung cancer (NSCLC) primary cells LSC1 (Fig. IGF1 under different dosages (50 ng, 400 ng, 2 μg, and 10 μgper 1 A and B). Synthetically, one-third (4/12) of lung cancer PDXs mouse) and found that tumor recurrence could only be induced at could initiate the suspensive