Formation of contacts between HB1.F3 NSCs and glioma cells in vivo is a complex process involving both the tumor cells and their interactions with surrounding brain and tumor stroma to form an overall microenvironment. The experiments reported here were designed to investigate formation of contacts between HB1.F3 NSCs and target cells in a peptide hydrogel matrix in which contributions from the microenvironment are minimized, thereby allowing analysis of cell behaviors in relative isolation. We evaluated HB1.F3.eGFP contact and encirclement of target glioma and non-glioma tumor cell lines, a non-malignant cell line, patient-derived glioma cells, primary mouse and human astroglial cells, and primary human dermal fibroblasts. Our major conclusion is that outside of the brain, in the absence of any additional external cues, HB1.F3 NSC contact and subsequent encirclement of glioma cells and other non-self target cells appears to be an expression of intrinsic behaviors that are not directed at tumor cells per se. As illustrated in , quantitative comparisons of HB1.F3 NSC contacts with target cells did not reveal significant preferences (with the notable exception of self-encirclement), indicating that many cell surfaces are permissive for contact formation. These observations therefore suggest that preferential formation of cell contacts in vivo (as well as long range migration which is not addressed in this study) involves contributions originating in the microenvironment created by tumor cells and surrounding brain.
It is important to note that while migratory behaviors of HB1.F3 cells appear to be cell-intrinsic, they are also quite plastic. Comparing 2- and 3-dimensional culture conditions, the differences between movements with a pattern of extension followed by soma translocation extension of HB1.F3 cells in the 3-dimensional Puramatrix environment described here and the predominately amoeboid movements exhibited by these same cells in 2-dimensional culture 
for other examples) point to considerable sensitivity of these NSCs to variations in surrounding environmental cues. This flexibility in choice of migratory mechanisms could be reflected in the two distinct phases of HB1.F3 NSC movements observed here: locomotion in Puramatrix and encirclement of target cells. We noted that the intermittent movements of HB1.F3 cells in Puramatrix were reminiscent of neural progenitor and immature neuron migration during cortical development 
, as well as to glial precursor movements and glioma cell dissemination 
. While differing in mechanistic detail, cell movements in all these systems show a thematically common pattern of leading process extension followed by soma translocation. Subsequent formation of target cell contacts by HB1.F3 cells and initiation of encirclement appear to evoke a mechanistic program in which F-actin and myosin II interact to drive lamellipodia expansion, as in other instances of cell migration 
. This transition from one migratory program to another in response to a change in the substrate 
may be reflected in differential sensitivity to cell-surface signals that for HB1.F3::HB1.F3 interactions allow for formation of homotypic contacts that are not permissive for subsequent extension of lamellipodia.
Some tumor-tropic stem cells imaged in vivo
in fixed tissue, including HB1.F3 NSCs, immortalized rat neural progenitor cells, mouse bone marrow-derived NSCs, mouse primary NSCs, and endogenous mouse neural precursor cells, appear adhered to tumor cells in preference to other brain cells 
. One possible model would have NSCs relatively immobilized once in tumor cell contact. However, these static images are only snapshots of dynamic processes, and they give no indication of the temporal stability of NSC-tumor cell contacts. In an alternative view, NSCs could be in constant motion, moving over a variety of permissive substrates during migration towards tumor sites. In this scenario, signals originating in the tumor microenvironment, in conjunction with tumor cell-derived signals, could stabilize an NSC swarm in and around tumor foci. Either way, the observations presented here point towards extensive engagement of NSCs with the tumor microenvironment, the element missing in these hydrogel-based cultures in promoting selective contacts between NSCs and tumor cells. Cell surface components within tumor cell niches, including extracellular matrix (ECM) associated with vasculature and axon tracts, soluble signals originating in the brain parenchyma stimulated by the presence of tumor cells or tumor stroma, along with the plethora of other reciprocal tumor-brain interactions established at tumor foci, appear in the aggregate to create a microenvironment favoring HB1.F3 proximity to glioma cells.
It has been suggested that essentially all glioma cells will have to be eliminated will be required to achieve curative efficacy in patients 
, and given the disseminated nature of high-grade glioma, cell-based therapies incorporating autonomous tracking of tumor cells are a promising strategy. Therefore, understanding the details of therapeutic and target cell interactions will be important for optimizing therapeutic designs. The investigation presented here separates processes of contact between NSCs and tumor cells from their brain milieu, in an effort toward this goal for NSC-based cancer therapies 
. The properties of HB1.F3 NSCs and their therapeutic potential have been studied to a greater extent than other NSCs. In summary tables of experimental investigations of potential NSC-based therapies, 5 of 13 studies compiled by Ahmed et al. 
and 10 of 18 listed by Kim 
involve HB1, F3 cells, and, as noted, they are currently in clinical trial 
. Because of their advanced development as therapeutic cells, understanding details of cell-cell interactions by HB1.F3 NSCs has the potential to directly further clinical development by, for example, manipulation of their intrinsic properties. An example of how this might occur is provided by Jurvansuu et al. 
, who demonstrated that up-regulation of cytokine receptor expression in NSC lines enhanced migration efficiency.
More generally, it has been widely noted that migration towards brain tumors is a property shared by many types of stem cells, including adult, neural progenitor and embryonic stem cell-derived NSCs 
(for reviews see 
) and mesenchymal stem cells (MSCs) of multiple origins 
(for reviews see 
). While there have been few direct comparisons of potentially therapeutic NSCs and MSCs 
, differences in migration and other behaviors may exist as consequences of underlying signaling pathways that will vary between stem cells of different lineages. For example, subtle distinctions have been noted between embryonic stem cell-derived and somatic NSCs in differentiation potential and proliferation 
, and MSCs of different origins differ in proliferation, differentiation potential and tumor-homing capabilities 
. In addition, a difference in the capacity of NSC and MSC lines to deliver a therapeutic oncolytic adenovirus payload has been reported 
. Ultimately, these variations in the specific properties of particular therapeutic NSCs, MSCs or other cells will impact treatment efficacy and therefore influence the design of these stem cell-based therapies.