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Supplementary Figures S1-S17 from Dynamic Glycoprotein Hyposialylation Promotes Chemotherapy Evasion and Metastatic Seeding of Quiescent Circulating Tumor Cell Clusters in Breast Cancer

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posted on 2023-09-06, 17:32 authored by Nurmaa K. Dashzeveg, Yuzhi Jia, Youbin Zhang, Lorenzo Gerratana, Priyam Patel, Asif Shajahan, Tsogbadrakh Dandar, Erika K. Ramos, Hannah F. Almubarak, Valery Adorno-Cruz, Rokana Taftaf, Emma J. Schuster, David Scholten, Michael T. Sokolowski, Carolina Reduzzi, Lamiaa El-Shennawy, Andrew D. Hoffmann, Maroua Manai, Qiang Zhang, Paolo D'Amico, Parastoo Azadi, Karen J. Colley, Leonidas C. Platanias, Ami N. Shah, William J. Gradishar, Massimo Cristofanilli, William A. Muller, Brian A. Cobb, Huiping Liu

Supplementary Figure S1-S17 show the methods of CTC analysis and CTC-associated survival (S1); glycosylation profiles of patient CTCs (S2); phenotypic effects of neuraminidase treatment and CD44 depletion on tumor clustering and sialylation (S3); N-glycomic analysis of α2,6-SA peaks in WT and ST6KO cells (S4); Depletion of ST6GAL1 or ST3GAL1 promotes clustering and chemo-evasion (S5); ST6GAL1 expression and cell sensitivity to therapeutics (paclitaxel, doxorubicine and palbociclib) (S6); he altered gene pathways in ST6GAL1-KO cells (S7); ST6GAL1 is associated with cell growth and patient survival (S8); dynamic glycosylation profile in PDXs and breast tumors (S9); ST6GAL1 suppresses metastatic seeding (S10); Counts and Ki67 expression of CTCs and DTCs in situ (S11); depletion of ST6GAL1 promotes transendothelial migration and seeding (S12); α2,6-sialylation of CD44 by ST6GAL1 (S13); ST6GAL1 substrates regulate cluster formation via altered binding affinity (S14); the down-regulation of ST6GAL1 substrates inhibits metastatic seeding (S15); anti-PODXL inhibits tumor cell cluster formation and metastatic seeding (S16); and anti-PODXL effects on CTCs and seeding of PDX-M1 (S17).

Funding

National Cancer Institute (NCI)

United States Department of Health and Human Services

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National Institute of General Medical Sciences (NIGMS)

United States Department of Health and Human Services

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NIH Office of the Director (OD)

American Cancer Society (ACS)

Susan G. Komen (SGK)

Lynn Sage Cancer Research Foundation (LSCRF)

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ARTICLE ABSTRACT

Most circulating tumor cells (CTC) are detected as single cells, whereas a small proportion of CTCs in multicellular clusters with stemness properties possess 20- to 100-times higher metastatic propensity than the single cells. Here we report that CTC dynamics in both singles and clusters in response to therapies predict overall survival for breast cancer. Chemotherapy-evasive CTC clusters are relatively quiescent with a specific loss of ST6GAL1-catalyzed α2,6-sialylation in glycoproteins. Dynamic hyposialylation in CTCs or deficiency of ST6GAL1 promotes cluster formation for metastatic seeding and enables cellular quiescence to evade paclitaxel treatment in breast cancer. Glycoproteomic analysis reveals newly identified protein substrates of ST6GAL1, such as adhesion or stemness markers PODXL, ICAM1, ECE1, ALCAM1, CD97, and CD44, contributing to CTC clustering (aggregation) and metastatic seeding. As a proof of concept, neutralizing antibodies against one newly identified contributor, PODXL, inhibit CTC cluster formation and lung metastasis associated with paclitaxel treatment for triple-negative breast cancer. This study discovers that dynamic loss of terminal sialylation in glycoproteins of CTC clusters contributes to the fate of cellular dormancy, advantageous evasion to chemotherapy, and enhanced metastatic seeding. It identifies PODXL as a glycoprotein substrate of ST6GAL1 and a candidate target to counter chemoevasion-associated metastasis of quiescent tumor cells.This article is featured in Selected Articles from This Issue, p. 1949

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