In this article, we describe a unique response of MM cells to mechanical perturbation, manifested in the formation of long tubular protrusions, which we termed FLIPs. The formation of FLIPs is a shear stress-induced process, which appears to be specific to MM cells exposed to such stress (shown here for 3 separate MM cell lines), and was not observed in any of the other adherent cell types tested, nor in MM cells in stationary cultures. Notably, besides the formation of robust FLIPs, the MM cells stimulated by shear force underwent a major structural reorganization, manifested in their alignment in the direction of flow, and loss of filopodia and lamellipodia from the entire cell body.
Extracellular stimuli may induce membrane protrusions in various cell types, often associated with diverse cellular functions. For example, macrophages contact their targets by actively extending actin-rich protrusions, depending on Rac activity (Flannagan et al., 2010
); Fusion-competent myoblasts form multiple finger-like extensions which invade neighboring muscle founder cells during myoblast fusion in Drosophila
(Sens et al., 2010
); extracellular matrix and soluble factors (e.g., EGF) can induce filopodial and lamellipodial protrusions in various cell types (Hu et al., 2010
; Mori et al., 2010
). While these serve as examples of membrane modification by extracellular biochemical signals, biophysical cues may also trigger the formation of membrane protrusions, including the extension of FLIPs, as described in the current study.
While FLIP formation is a novel phenomenon, cellular responses to shear flow have been documented in diverse cell types. It has been shown that endothelial cells subjected to near-physiological shear undergo uniform alignment (Dewey et al., 1981
; Galbraith et al., 1998
; Masuda and Fujiwara, 1993
), and directional migration and lamellipodial extension in the direction of flow (Dewey et al., 1981
; Wojciak-Stothard and Ridley, 2003
; Zaidel-Bar et al., 2005
). Hematopoietic cells, which reside, at least transiently, in high-shear vascular environments, respond to flow in a variety of ways. T cells undergo dynamic shape changes during trans-endothelial migration, including tethering and rolling along the endothelial surfaces, firm attachment to the underlying cells, spreading on them, and trans-migration through the endothelial cell layer (Alon and Dustin, 2007
; Dong et al., 1999
; Stroka and Aranda-Espinoza, 2010
). Platelets also go through several shape changes, including transition from a round morphology, forming multiple elongated extensions during the adhesive process under flow (Kuwahara et al., 2002
). In addition, flow-induced effects were seen in other cell types, such as rolling of human bone-metastatic prostate tumor cells on endothelial cells (Dimitroff et al., 2004
); transendothelial migration of melanoma cells (Slattery and Dong, 2003
); increased adhesion and spreading in colon cancer cells (Burdick et al., 2003
; Kitayama et al., 1999
); and elongation and reorientation in osteoblasts (Liu et al., 2010
). In all these cases (as well as in the present work), the response to shear flow was apparent, yet the exact cellular site where the mechanical perturbation is being sensed (e.g., dorsal cell surface, adhesion sites to the matrix) remains unclear (Bershadsky et al., 2003
; Cao et al., 1998
; Chen, 2008
Similar to these shear-dependent processes, FLIP formation appears to be an active process, triggered by external force and driven by the cytoskeleton. This notion is supported by the abundance of actin filaments in the FLIP, and its tendency to undergo extension-retraction cycles under constant shear.
An interesting feature of FLIPs is the tight correlation between the amount of force applied, and both the number of FLIP-forming cells (ranging from ~5% under 4 dynes/cm2, to ~35% under 28–36 dynes/cm2) and the time interval between the application of force, and the average onset of FLIP extension (9 min for 12 dynes/cm2, and 2.68 min for 36 dynes/cm2). Time-lapse monitoring of the affected cells confirmed that different cells within the MM cell population exhibit different mechanosensing thresholds, affecting the extent and rates of FLIP formation. This increase in the number of FLIPs under strong shear is attributed to an increase in the numbers of mechano-responsive cells, while the rates of FLIP retraction, and the average lifespan of the FLIPs, remained unchanged under different levels of shear stimulation ().
An additional characteristic feature of the response of the cells to high-shear stimulation is the apparent adaptation of the cells to the flow, manifested in a decrease in the number of FLIP-forming cells, following lengthy exposure (about 30 minutes) to the flow. This finding indicates that shear-induced FLIP formation can be down-regulated by the cells, possibly by modulation of the mechanical threshold levels. Single-cell analysis further demonstrated the heterogeneity of the cellular response to force. Increased shear forces resulted in earlier and more homogenous FLIP onset times, while under weaker forces, FLIP onset was more variable. Furthermore, the cell population could be roughly divided into two subpopulations, based upon FLIP onset and duration time: a shorter FLIP lifetime was correlated with lower sensitivity to force, as more of these cells responded later to its application, while cells demonstrating longer FLIP lifetimes responded more quickly (). It is possible that variations in sensitivity to force reflect different potential adhesion sites in different cells, corresponding to the various forces encountered in their path and, in that manner, enabling each of the cells’ subpopulations to adhere or extravasate at diverse locations.
The studies described above reveal the intriguing biophysical characteristics of FLIP formation, retraction and long-term regulation; yet the physiological relevance of this structure remains elusive. In this work, we have considered several potential processes relevant to MM cell physiology that could, potentially, be relevant to FLIP formation. MM cells develop in the BM; yet they are capable of exiting the BM via the vascular system, and then home back to BM niches throughout the body (Van Camp and Van Riet, 1998
; Vande Broek et al., 2008
). Thus, the process of disease dissemination includes obligatory intravasation and extravasation events, which involve major transitions in the hydrodynamic characteristics of the cellular environment. These include a transition from the relatively sessile BM niche, to the BM vasculature, to a high shear environment in the major blood vessels, ranging from 1–10 dynes/cm2
in venules, to 60 dynes/cm2
and more in arterioles (Slack and Turitto, 1993
). It is noteworthy that this range of shear forces is within the range that was found here to trigger FLIP formation in different populations of MM cells. Interestingly, despite showing migratory capabilities in stationary cultures, ARH-77 type A cells did not appear to migrate during exposure to flow, regardless of FLIP formation or retraction. The extravasation step appears to be the most challenging one, requiring firm adhesion of the cells to the vessel wall (to withstand the shear drag forces), and migration through it. One attractive possibility that we considered was that the FLIPs produced might be instrumental in triggering a transendothelial migration process. To test this possibility, we exposed a monolayer of endothelial cells with ARH-77 cells attached to it, to shear flow, and monitored the fate and dynamics of the attached cells. As shown in Supplementary Figure 1
, above, we could detect FLIP formation in such stressed, mixed cultures, as well as penetration of the MM cells into the gap between the endothelial cells and the substrate; however, we found no evidence that this transendothelial migration was particularly enhanced in FLIP-forming cells, nor that shear flow affected this process. Another hypothesis is that FLIP formation might decrease cellular resistance to flow and thus reduce the drag forces acting on the cells, thereby increasing their retention on the vessel wall. A rough calculation of the expected flow resistance suggested that a hemispherical MM cell, covered by multiple filopodial and lamellopodial protrusions and typically several micrometers in length (as in cells residing in a stationary environment), is subjected to severalfold-higher drag forces, compared to an aligned FLIP-forming cell of the same volume. Given that MM cells display a wide range of “mechanosensitivity thresholds,” it appears possible that different cells might preferentially form FLIPs, and develop more stable adhesions with blood vessels exposed to distinct levels of shear stress.
In recent years, it was established that the microenvironment plays a critical role in the development of B-cell malignancies. Most studies in the field point to interactions between MM cells with cellular constituents of the microenvironment (e.g., osteoclasts, mesenchymal stromal cells) as key factors controlling the survival and proliferation of the transformed cells. Moreover, some clinical manifestations of the disease are facilitated by physical MM-microenvironment interactions; for example, the generation of lytic bone lesions by MM-osteoclast co-aggregates (Terpos et al., 2009
), and the stimulation of SDF1/CXCR4-mediated cell migration (Katz, 2010
; Zhang et al., 2009
Shear stress is undoubtedly an important constituent of the microenvironment of migrating MM cells; yet no information is available with respect to its effect on these cells. Here we show, for the first time, that some MM cells are equipped with mechanism(s) to sense and respond to forces exerted by microenvironmental shear stress. Moreover, we found that different variables of the shear flow, including a wide range of forces and duration of the flow, are within the “detection capabilities” of these cells. Finally, the data described here reveal profound diversitywithin the MM cell population, with respect to mechanosensory responsiveness.