(A-26) Investigation of integrin-α2 in breast cancer leader cells

Introduction:: Collective migration, the movement of clusters of cells together, is the main mode of migration by which breast tumor cells metastasize in vivo [1]. During collective migration, cancer cells migrate in a cohesive, multicellular unit, led by a subgroup of cells, known as leader cells, located at the front edge. Work in our lab and others found that leader cells are positive for cytokeratin-14 (K14) during breast cancer collective migration [2]–[4]. While we know leader cells sense and interact with their environment to direct migration, the mechanisms and the specific receptors involved with this process is not fully understood. One family of mechanoreceptors used by cells to interact with the extracellular matrix (ECM) is integrins. Integrins are linked to actin filaments through focal adhesion proteins, thus affecting cell contractility and migration [5]. One integrin that has been identified as a key regulator of adhesion, motility, and invasiveness in cancer cells is integrin-α2 (ITGα2) [6]. Blockage of ITGα2 in murine models of breast cancer resulted in reduced liver metastasis [7]. ITGα2, which binds to integrin-β1 (ITGβ1), senses collagen 1 and translates signal to activate intracellular migration signaling pathways [8]. The presence or function of ITGα2 in breast tumor leader cells is not known. Therefore, our objective is to investigate the presence and role of ITGα2 in breast tumor leader cells. Our hypothesis is that breast tumor leader cells express higher levels of ITGα2 than follower cells, which is used to sense mechanical cues from the ECM to initiate collective migration.


Materials and Methods:: First, we wanted to understand which integrins are critical for ECM sensing in collective migration. In prior studies, we generated aligned and random collagen hydrogels, cultured primary MMTV-PyMT tumor cell clusters (organoids) on them, and found that organoids preferentially spread out on aligned collagen hydrogels, a form of collective migration. After culturing tumor cell clusters on aligned and random hydrogels for 3-6 days, we isolated cells from the matrix using chloroform and Trizol. We lysed cells, extracted mRNA (Qiagen) and converted to cDNA (Applied Biosystems™). We performed qRT-PCR to quantify gene expression using Taqman probes for ITGα2, ITGα6, ITGβ1 and ITGβ4 (QuantStudio 3 System) (Fig 1). Next, we validated our qRT-PCR results using immunostaining (Fig 2A-B). Tumor organoids were seeded onto fibronectin and collagen 1 coated PDMS coverslips and incubated (24 hr, 37°C). Organoids were fixed, blocked, and stained for cytokeratin-14 (K-14; leader cell marker), vinculin (focal adhesion), integrin-β1, ITGα2 and activated integrin-β1 (9EG7). Alexa Fluor™ secondary antibodies were species matched, and organoids were counterstained for DAPI. Samples were imaged via confocal microscopy (Zeiss LSM 980) at 63x air 1.0 zoom with slices taken every 1 μm to obtain a z-stack. Colocalization analysis was performed using FIJI plugin, Coloc2.

Results, Conclusions, and Discussions:: Results & Discussion: qRT-PCR results reveal increased gene expression of ITGα2 and ITGβ1 in organoids cultured on aligned collagen hydrogels compared to random collagen hydrogels (Fig 1). Through immunofluorescence staining, we observed positive expression of ITGα2 in the K14+ leader cells, but not the K14- follower cells (Fig 2A). This was confirmed through Pearson’s colocalization analysis which revealed a strong positive correlation between ITGα2 and K14 staining intensities (Fig 2A). ITGβ1 was ubiquitous on all cells (data not shown), but there was positive staining for activated ITGβ1 (9EG7) at the leading edges (Fig 2B) which is consistent with previous studies demonstrating activated ITGβ1 is highly localized in the lamellipodia of leader cells [9]. Further, we observed more vinculin (focal adhesion protein) in leader cells compared to follower cells, suggesting the forces exerted by leader cells may be greater than follower cells. Altogether, these results begin to suggest that activated ITGβ1, ITGα2, and vinculin may have a unique function in K14+ leader cells associated with migration. Our results correlate with other published studies that also demonstrate unique gene and protein expression of ITGα2 in leader cells that is absent in follower cells via genetic analysis [10].

Conclusion: Our findings demonstrate higher expression of ITGα2 in leader cells compared to follower cells, which suggest leader cells use ITGα2 as part of their mechanosensing machinery to sense environment cues and drive migration. In future work we will investigate the functional role of ITGa2 through selective knockdown of ITGα2 in leader cells. Understanding how leader cells mechanically respond during collective migration could provide new targets for therapy design to prevent or treat metastasis.


Acknowledgements (Optional): : Thanks to Jinkal Modi and Dr. Anjan Pradhan for assistance with qRT-PCR. Thanks to the Nanocharacterization Core at VCU for microscopy help.


References (Optional): : [1] A. N. Giaquinto et al., “Breast Cancer Statistics, 2022,” CA: A Cancer Journal for Clinicians, vol. 72, no. 6, pp. 524–541, 2022, doi: 10.3322/caac.21754.

[2] K.-V. Nguyen-Ngoc et al., “ECM microenvironment regulates collective migration and local dissemination in normal and malignant mammary epithelium,” PNAS, vol. 109, no. 39, pp. E2595–E2604, Sep. 2012, doi: 10.1073/pnas.1212834109.

[3] K. J. Cheung et al., “Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumor cell clusters,” Proc. Natl. Acad. Sci. U.S.A., vol. 113, no. 7, pp. E854-863, Feb. 2016, doi: 10.1073/pnas.1508541113.

[4] P. Y. Hwang, A. Brenot, A. C. King, G. D. Longmore, and S. C. George, “Randomly Distributed K14+ Breast Tumor Cells Polarize to the Leading Edge and Guide Collective Migration in Response to Chemical and Mechanical Environmental Cues,” Cancer Res, vol. 79, no. 8, pp. 1899–1912, Apr. 2019, doi: 10.1158/0008-5472.CAN-18-2828.

[5] Z. Sun, M. Costell, and R. Fässler, “Integrin activation by talin, kindlin and mechanical forces,” Nat Cell Biol, vol. 21, no. 1, Art. no. 1, Jan. 2019, doi: 10.1038/s41556-018-0234-9.

[6] Y. Matsumoto et al., “Integrin alpha 2 is associated with tumor progression and postoperative recurrence in non-small cell lung cancer,” Jpn J Clin Oncol, vol. 53, no. 1, pp. 63–73, Jan. 2023, doi: 10.1093/jjco/hyac148.

[7] V. Adorno-Cruz and H. Liu, “Regulation and functions of integrin α2 in cell adhesion and disease,” Genes Dis, vol. 6, no. 1, pp. 16–24, Dec. 2018, doi: 10.1016/j.gendis.2018.12.003.

[8] E.-S. Kim et al., “C-reactive protein binds to integrin α2 and Fcγ receptor I, leading to breast cell adhesion and breast cancer progression,” Oncogene, vol. 37, no. 1, pp. 28–38, Jan. 2018, doi: 10.1038/onc.2017.298.

[9] N. Yamaguchi, T. Mizutani, K. Kawabata, and H. Haga, “Leader cells regulate collective cell migration via Rac activation in the downstream signaling of integrin β1 and PI3K,” Sci Rep, vol. 5, no. 1, Art. no. 1, Jan. 2015, doi: 10.1038/srep07656.

[10] B. Chen, Y. Tang, Y. Tang, and X. Liang, “What makes cells move: Requirements and obstacles for leader cells in collective invasion,” Experimental Cell Research, vol. 382, no. 2, p. 111481, Sep. 2019, doi: 10.1016/j.yexcr.2019.06.026.