Targeting the reattachment of circulating breast tumor stem cells to reduce metastasis. Stuart S. Martin, Ph.D. Associate Professor of Physiology University of Maryland School of Medicine The limits of clinical imaging shape our understanding of cancer recurrence Primary Tumor Circulating Tumor Cells (CTCs) Seeding Dormancy Below clinical detection threshold PET/CT MRI Metastatic Recurrence Foci > 10 million tumor cells PET/CT MRI Dynamic CTC responses Eradicate these cells Delay or Eliminate Recurrence “Dormant” cells produce dynamic membrane protrusions when detached MCF10A mammary epithelial cells Frame / 1 sec. Actin rearrangement in MCF10A cells attached to extracellular matrix Cytoskeletal support of membrane protrusions Rebecca Bettes et al., Exp. Cell Res. 313:1326-36 (2007). Control LA Inhibit actin polymerization Destroys Filopodia and invadopodia Phase Contrast α-tubulin Microtentacles (McTN) Actin Microtentacles promote tumor cell aggregation (Live confocal imaging) Rebecca Bettes et al., Exp. Cell Res. 313:1326-36 (2007). Microtentacles increase in invasive/metastatic breast tumor cell lines Rebecca Whipple et al., Cancer Research 68:5678-5688 (2008). Strongly Metastatic Weakly Metastatic In vivo mechanism for circulating tumor cell attachment matches microtentacles Korb et al., Exp Cell Res 2004; 299: 236-247. adherent Tubulin required for attachment HT-29 colon carcinoma cells CalceinAM labeled Injected into portal vein Imaged in liver sinusoidal capillaries Actin depolymerization enhances attachment Confocal imaging of live tumor cell attachment to endothelial cells Primary tumor Death from fragmentation Inhibit microtentacles Fates of circulating cells? Death from apoptosis [1] ADHESION [2] EXTRAVASATION Actin-dependent Microtentacles Metastatic tumor Current working model of microtentacle (McTN) structure Glu-tubulin microtubules MTOC TTL Unidentified Carboxypeptidase (TCP) Actin cortex Tyr-tubulin microtubules Microtubule expansion is counteracted by actin cortical contraction EB1-GFP imaging ECM-attached cell Detached cell Kinesin inhibitors reduce microtentacles Jennifer Yoon et al., Breast Cancer Research and Treatment (2011) Tetracaine (125µM) EMT induces microtubule detyrosination and microtubules Rebecca Whipple et al., (Cancer Research, 2010) Vector Twist Snail Attached E-Cad N-Cad Vim Twist 250μm Suspended Snail 10μm 50 Snail Actin 10 2% 0 Vector Twist Snail Snail 20 TTL 30% Twist (% cells) α-tub Vector Ctl 30 Glu 41% 40 McTNs Twist Vector Ctl β-actin McTNs mediate tumor cell reattachment (matrix/endothelial) 3 Twist Snail GFP 2.5 Cell Index 2 1.5 1 0.5 0 0 5 15 25 35 45 55 65 Time (min) 100% * * TWIST SNAIL % Attachment 80% 60% 40% 20% 0% GFP Concurrent upregulation of Twist and Glu-tubulin at invasive tumor fronts Rebecca Whipple et al., (Cancer Research, 2010) 100x 200x 200x Microtentacles are increased in cancer stem cell populations Charpentier, M. et al. Cancer Research (2014) Stem cell subpopulations reattach more efficiently Charpentier, M. et al. Cancer Research (2014) Microtentacles are observed in mammospheres Charpentier, M. et al. Cancer Research (2014) Microtentacles extend between cells in mammospheres Charpentier, M. et al. Cancer Research (2014) Drugs targeting cell division can enhance microtentacles Eric Balzer et al., Breast Cancer Research and Treatment (2010) McTN(%) Jas +Taxol Jas Taxol Vehicle Tumor Growth Jas + Paclitaxel Vehicle CTC Metastasis? Parthenolide and Costunolide can selectively target Glu-tubulin Rebecca Whipple et al., (Breast Cancer Research, 2013) Vehicle Glu-tubulin α-tubulin Parthenolide Parthenolide and Costunolide inhibit tumor cell reattachment Rebecca Whipple et al., (Breast Cancer Research, 2013) Epithelial Sheet Wound Edge Free-floating Weak MT Strong MT Strong MT Strong Actin Weak Actin Weak Actin Growth EMT, Growth Stemness MT Actin Microtentacles Microtubule Stabilizers (Paclitaxel, Ixabepilone) Strong MT Stemness? Actin inhibitors (Fasudil - ROCK, Dasatinib - Src) Weak Actin Metastasis? Chemotherapy before surgery and CTC levels (from Hekimian et al., 2012) Cumulative relapse-free survival 1 0.8 0.6 Neoadjuvant therapy 0.4 CTCs decrease CTCs increase 0.2 0 0 2 Years 4 6 8 Surgical samples from breast cancer patients (membrane dynamics) Patent pending Surgical samples from breast cancer patients (membrane dynamics – 600x) Patent pending Drug responses can be measured quickly in patient-derived tumor cells (30 minute Colchicine response) Patent pending Automated measurement of McTN characteristics A B Confocal imaging of patient tumorgraft cells (HCI-001) Automated McTN Analysis (MATLAB) Red: >4µm McTN length Green: <4µm McTN length #McTNs/cell 11 4 6 Avg. McTN length (µm) 8.8 10.2 8.7 HCI-001 cell population McTN frequency = 34% Avg.# McTNs/cell = 9.0 Avg. McTN length = 9.1µm Patent pending Department of Physiology Rebecca Whipple Bettes Eric Balzer Agnes Cheung Jennifer Yoon Ed Cho Mike Matrone Michele Vitolo Keyata Thompson Monica Charpentier Amanda Boggs Susette Mueller (Georgetown) Jing Yang (UCSD) Josef Kas (Leipzig) UM-College Park Wolfgang Losert Eleanor Ory Ben Shapiro Zach Cummins R01-CA124704, R01-CA154624 (NCI) Era of Hope Scholar Award (Department of Defense) Susan G. Komen for the Cure – KG100240 Department of Defense Breast Cancer Idea Award Department of Defense Breast Cancer Concept Award DOD Breast Cancer Predoctoral (Cho) DOD Breast Cancer Predoctoral (Balzer) ACEA xCelligence S10-RR022434-01 (NCRR, Xenogen) ACEA Travel Award FAMRI Clinical Innovator Award UMB Independent New Investigator Award K01-CA096555 Howard Temin Career Award (NCI) Maryland Stem Cell Research Foundation Maryland Cigarette Restitution Fund
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