Cancer Letters

Cancer Letters

Volume 506, 28 May 2021, Pages 142-151
Cancer Letters

Physical confinement during cancer cell migration triggers therapeutic resistance and cancer stem cell-like behavior

https://doi.org/10.1016/j.canlet.2021.01.020Get rights and content

Highlights

  • Tight confinement during cancer cell migration triggers therapeutic resistance and induces cancer stem cell-like behavior.

  • Increasing efflux proteins and cancer stem cell markers associate with increased therapeutic resistance.

  • Re-localization of YAP to the cell nucleus indicates an elevated level of cytoskeletal tension during confined-migration.

Abstract

Metastasized cancer cells have an increased resistance to therapies leading to a drastic decrease in patient survival rates. However, our understanding of the cause for this enhanced resistance is lacking. In this study, we report that physically tight confinement during cancer cell migration triggers therapeutic resistance and induces cancer stem cell-like behavior including up-regulation in efflux proteins and in cancer stem cell related markers. Moreover, the re-localization of Yes-associated protein (YAP) to the cell nucleus indicated an elevated level of cytoskeletal tension. The increased cytoskeletal tension suggested that mechanical interactions between cancer cells and tight surroundings during metastasis is one of the factors that contributes to therapeutic resistance and acquisition of cancer stem cell (CSC) like features. With this system and supporting data, we are able to study cells with therapeutic resistance and CSC-like properties for the future purpose of developing new strategies for the treatment of metastatic cancer.

Introduction

More than 90% of mortality caused by cancer is attributable to metastases [1,2]. Due to its unique systemic nature and resistance to existing therapeutic agents, metastatic cancer is largely incurable with current common treatments [1,3].

Cancer cell migration is a key element of the invasion-metastasis cascade; the extraordinary motility and deformability of metastatic migrating cells allow them to easily move through physical confinement [4,5]. During this process, cells undergo a change known as metastatic resistance [6]. Although the major mechanisms of resistance have been widely investigated, the relationship between migration in physical confinement and the procurement of therapeutic resistance is not well elucidated. In addition, a rare population of tumor cells with stem cell-like properties, labeled as cancer stem cells (CSCs), can play a distinct role in driving metastasis. CSCs are difficult to study because of their small number within the tumor population. Previous works have suggested that CSC initiation and maintenance may be related to physical inputs such as extracellular matrix stiffness within the local microenvironments [7,8]. Hence, mechanical inputs such as interstitial pressure from the microenvironment, might greatly affect the CSC-like behaviors of cancer cells and drive metastatic invasion. A critical step to improve our understanding of the mechanisms underlying treatment resistance is to generate appropriate tools with physical inputs to induce or enrich for treatment resistant cancer cells.

In this study, we produced a group of confined-migrating cancer cells by allowing them to migrate in a physically confined microenvironment (i.e., a microchannel with a width that is smaller than the cells size); and carried out detailed molecular analyses on those confined-migrating cancer cells to establish the relationship between mechanical interaction and acquired therapeutic resistance. This study focuses on G55 glioblastoma and MDA-MB-231 breast cancer cell lines. The aim of this study is to confirm that physical confinement induces chemotherapeutic and radio-therapeutic resistance. Detailed protein level comparison was used to determine which molecules are potential key players for physical confinement induced therapeutic resistance by Western blot analysis. Immunostaining and hypoxic analysis further allowed us to evaluate the relationship between cytoskeletal pressure, CSC-like properties and physical confinement induced therapeutic resistance. We also found a similar phenomenon (i.e., physical confinement induces therapeutic resistance) in lung, prostate, and patient-derived GBM. Thus, we believe this physical confinement induces therapeutic resistance as a universal trait in various cancer types.

Section snippets

Fabrication of microchannel devices

Two different styles of microchannel devices described in our previous studies were used for this study (demonstrated in supplemental information Fig. S1. A and B) [9]. Combined utilization of standard negative photolithography and soft lithography was the core of our microchannel device fabrication, with recessed features fabricated on a silicon wafer coated with SU-8 photoresist (Microchem Corp, Newton, MA). The thickness of coating was controlled by spin speed which determined the

Confined-migrating cancer cells develop chemotherapy and radiotherapy resistance

The confined-migrated cells moving through physical confinement showed characteristic resistance to chemo-therapeutic agent, Doxorubicin (Dox). Both GBM and MDA-MB-231 cancer cell lines were treated with 17 μM Dox for visualization of intracellular drug mobility, due to the degree of Dox autofluorescence at this concentration [11]. After 4 h Dox incubation, samples were imaged at two different timepoints: immediately after Dox removal (4hr) and 16 h (overnight). The autofluorescence of Dox in

Discussion

Currently, treatment with radiation and chemotherapy after surgical resection to remove tumors is the standard-of-care for many cancer patients [4,26]. However, the highly therapeutic resistant nature of metastatic cancer cells tends to limit the efficacy of current cancer treatments. A better understanding of what triggers confined-migration (via physically constricted tissue) induced resistance is important for improving anticancer strategies, specifically for metastatic cancer. In this

Funding

This work was supported by Cancer Prevention and Research Institute of Texas (RP150711) and Institutional Development Award from the National Institute of General Medical Sciences of the National Institutes of Health (P20GM103639).

Author contributions

Y.T.K. and J.D.B. were the principal investigators and conceived the idea. Q.S., T.H. and X.C. wrote the paper. Q.S. and T.H. developed the idea and designed the experiments. Y.T.K. and L.B. designed the microchannel devices. Q.S. provided all G55 data. T.H. provided all MDA-MB-231 data. X.C. and J.D.B. carried out all radiation experiments. R.B. provided A549 data and E.H. provided PC3 data. All authors reviewed the manuscript and declared no competing financial interests.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank Dr. Kimberly Bowles, Dr. Kytai Nguyen, Dr. Yi Hong and Dr. Baohong Yuan for kindly providing us with training and facility access to conduct protein analysis. We thank Dr. Bo Chen for kindly assisted us with taking confocal images and Dr. Charles Chuong provided guidance for hydrodynamic simulation. We also thank Calvin Kong for reviewing the manuscript. Acknowledgement also goes to the University of Oklahoma Health Sciences Center for providing patient-derived cancer samples.

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