Bleb-based extravasation uses conserved morphodynamics but divergent calcium control

Domain: biology
Published: 2026-03-26
Editorial outcome: major_revision

Abstract
Extravasation, the exit of cells from blood vessels, is essential in both development and disease, from germ cell migration to cancer metastasis. We show that all extravasating cells examined, avian primordial germ cells and diverse human cancer lines, form Ca²⁺-dependent membrane blebs during transendothelial migration. Yet the source of Ca²⁺ diverges by lineage. PGCs and HT-1080 fibrosarcoma cells rely on store-operated Ca²⁺ entry (SOCE), whereas epithelial-derived cancer cells such as PC-3 and MDA-MB-231 use IP₃R-mediated Ca²⁺ release from the endoplasmic reticulum. These findings establish bleb-based extravasation as a conserved morphodynamic strategy powered by distinct regulatory modules. Evolutionarily, they map onto an ancestral ER-release pathway and a metazoan-derived SOCE pathway. Conceptually, cancer blebbing emerges as a composite strategy that reactivates both ancient survival programs and developmental toolkits to maximize invasive success. This unified framework highlights Ca²⁺ supply as a critical bottleneck of vascular escape, offering new angles for targeting metastatic dissemination.

Introduction
Extravasation, the exit of cells from blood vessels, is a fundamental process in both physiological and pathological contexts, including immune cell trafficking1, cancer metastasis2, microglial infiltration3, and germ cell migration4. In vertebrate embryos, primordial germ cells (PGCs) of avian species intravasate into the bloodstream and subsequently extravasate through the dorsal mesentery toward the gonadal region, where they initiate gametogenesis4,5. In metastasis, malignant cells exploit this same process to colonize distant tissues2. Despite its broad relevance, the morphodynamic and regulatory diversity of extravasation across lineages remains poorly understood.

Traditionally, extravasation has been attributed to actin-dependent protrusions such as lamellipodia and filopodia, which mediate endothelial transmigration through junctional openings6. We recently uncovered an alternative strategy: membrane blebbing. These balloon-like, pressure-driven protrusions arise from local detachment of the plasma membrane from cortical actin. Unlike lamellipodia, blebs support rapid, adhesion-independent migration and are broadly used across cell types, from amoeboid cells to mammalian immune cells7,8. This phenomenon was first observed in vivo during PGC extravasation in chick embryos4 and later extended to human cancer cells in the avian yolk sac vasculature (YSV)9. These observations provided the first in vivo evidence that both normal and malignant cells can deploy blebbing to breach the endothelium. Whether such protrusions are powered by a conserved molecular program or represent convergent solutions that culminate in similar morphologies remains unresolved.

Ca²⁺ signaling has been implicated in bleb formation across multiple biological contexts, including cell migration, cellular stress responses, and cell death10-15. Bleb-associated Ca²⁺ dynamics have been observed in a wide range of cell types, including primordial germ cells, cancer cells, hepatocytes, and cultured cell models such as HEK293 cells. In these systems, localized elevations of cytosolic Ca²⁺ are commonly detected during the early phase of bleb formation. Mechanistically, increases in cytosolic Ca²⁺ have been associated with enhanced cytoplasmic fluidity, which is proposed to promote rapid cytoplasmic flow into membrane protrusions during bleb expansion10,11. However, but whether this requirement represents a universal principle or diverges between lineages is unknown. Addressing this issue is essential, not only for defining the mechanistic logic of bleb-based migration, but also for testing whether cancer extravasation reflects co-option of developmental pathways or reactivation of more ancient cellular strategies.

Direct analysis in mammalian systems is limited by deep tissue location and rapid extravasation events. In contrast, avian embryos offer unique advantages: both the YSV and the PGC-specific extravasation vascular plexus (Ex-VaP) are planar, transparent, and accessible for single-cell in vivo imaging. Moreover, these models permit simultaneous transplantation of genetically distinct cells into a shared vascular environment4,9. This experimental platform provides an unprecedented opportunity to compare germ cells and cancer cells side by side, allowing us to dissect conserved principles and lineage-specific variations of bleb-based extravasation.

Here, we investigate the cellular dynamics and regulatory mechanisms of bleb-based extravasation using chick PGCs and human cancer cells. We show that both cell types undergo extravasation through conserved bleb-driven morphodynamics during transendothelial migration. However, the molecular regulation of this process diverges: while chick PGC extravasation critically depends on store-operated Ca²⁺ entry (SOCE), certain cancer cells extravasate independently of this pathway and instead rely on intracellular Ca²⁺ release mechanisms. These findings reveal that bleb-based extravasation represents a conserved physical strategy shared between developmental cell migration and cancer metastasis, while the upstream regulatory mechanisms have diverged between physiological and pathological contexts.

Results
Chick PGCs form membrane blebs during extravasation
To investigate the cellular dynamics of PGC extravasation, we focused on the chick Ex-VaP at Hamburger–Hamilton (HH) stage16 15, where circulating PGCs are selectively arrested prior to transendothelial migration (TEM). For live imaging, we used EGFP+ PGCs that had been expanded and genetically labeled in vitro, then reintroduced into the embryonic vasculature. Transplanted PGCs recapitulate the full migratory path to the gonads, faithfully mimicking their endogenous counterparts4, thus enabling high-resolution visualization under physiologically relevant conditions.

Live imaging of transplanted EGFP+ PGCs revealed two sequential protrusive behaviors. In the first phase, which we term intravascular crawling, cells rapidly produced multiple small, transient spherical protrusions (< 5 min) in random directions (Fig. 1a and Supplementary Movie1). This was followed by the emergence of a single, large, long-lived spherical protrusion (15–30 min) oriented toward the extravascular space, marking the onset of TEM (Fig. 1b and Supplementary Movie2).

To determine whether these protrusions are bona fide blebs, we examined cortical actin using Lifeact-mCherry. Both small and large protrusions showed transient loss of cortical actin (Fig. 1c,f), a hallmark of blebs10,17-19. During crawling, actin-rich protrusions such as filopodia were also observed, in addition to blebs (Fig. 1c). We next asked whether these blebs also display localized Ca²⁺ surges, a phenomenon frequently associated with motility-driven blebbing10,11. Imaging with GCaMP6s revealed transient Ca²⁺ increases precisely at protrusion sites (Fig. 1d,e,g,h). Thus, PGC blebs during extravasation are defined by the combined molecular signature of actin loss and localized Ca²⁺ elevation.

Importantly, similar bleb-like protrusions were also observed in endogenous PGCs undergoing TEM in vivo (Supplementary Fig. 1a), confirming that blebbing is an intrinsic behavior of chick PGCs rather than an artifact of culture or transplantation.

In vitro reconstitution reveals SOCE as a driver of bleb formation in chick PGCs
To dissect the mechanism of bleb formation in chick PGCs, we established a tractable in vitro system using under-agarose compression (UAC), a mechanical stimulus known to induce blebbing in diverse cell types20,21 (Supplementary Fig. 2a). Cultured PGCs reproducibly exhibited recurrent expansion and retraction of large spherical protrusions (Supplementary Fig. 2b), closely resembling their in vivo behavior. These protrusions coincided with localized Ca²⁺ surges and transient loss of cortical actomyosin, as visualized with GCaMP6s and Lifeact reporters (Supplementary Fig. 2c-g and Supplementary Movie3,4), consistent with bona fide membrane blebbing. Time-resolved analyses using kymograph representations further revealed that Ca²⁺ levels remain elevated throughout the bleb expansion–retraction cycle, whereas cortical F-actin accumulation temporally correlates with the retraction phase (Supplementary Fig. 2h).

Pharmacological perturbations implicated SOCE as the source of Ca²⁺. Chelation of extracellular Ca²⁺ with BAPTA or inhibition of endoplasmic reticulum (ER) Ca²⁺ release with 2-APB22,23, each strongly suppressed bleb formation (Fig. 2a,b). This dual requirement pointed to SOCE24,25, in which ER Ca²⁺ depletion triggers Orai Ca²⁺ channel opening at the plasma membrane (PM) via ER-resident STIM sensors that translocate to ER–PM contacts26,27.

Re-analysis of published transcriptomic data28 confirmed robust expression of STIM2 and Orai1/2 in chick PGCs at HH17 (E2.5), with STIM1 also detectable at low levels (Supplementary Table 1). Consistent with canonical SOCE architecture, fluorescently tagged STIM1 localized to the ER and Orai1 to the PM (Supplementary Fig. 3a,b). Notably, STIM1 signals were frequently detected within ER-ID–positive regions exhibiting relatively lower ER-ID intensity. ER-ID is a qualitative marker of overall endoplasmic reticulum distribution, and local variations in signal intensity cannot be interpreted as evidence for defined ER subdomains. Nevertheless, this observations indicate that STIM1 is not uniformly distributed throughout the ER but instead displays a biased localization under resting conditions in cultured PGCs. During bleb initiation and expansion, fluorescently tagged STIM1 and Orai1 dynamically co-localized at discrete ER–PM contacts within protrusions (Fig. 2c,d), consistent with localized SOCE activity11. To further assess whether STIM1 and Orai1 physically interact under bleb-inducing conditions, we employed a split-Venus complementation assay, in which STIM1 and Orai1 are fused to complementary fragments of Venus fluorescent protein. This system reports close molecular proximity between STIM1 and Orai1 through irreversible Venus fluorescence reconstitution. Under resting conditions, Venus fluorescence was not detected. In contrast, UA compression induced discrete Venus-positive puncta concomitant with blebbing (Fig. 2e), indicating stimulus-dependent STIM1–Orai1 complex formation.

Functionally, SOCE blockade with SKF96365 (hereafter SKF) abolished blebbing (Fig. 2a,b). Moreover, treatment with BTP2, a more selective inhibitor of STIM-dependent SOCE, completely eliminated detectable bleb formation (Fig. 2a,b). These results indicate that SOCE activity is required, at least for bleb initiation, in PGCs. In contrast, ER Ca²⁺ depletion with thapsigargin—normally sufficient to activate SOCE—robustly induced bleb formation even without mechanical stimulation (Fig. 2f). Under this thapsigargin-induced condition, STIM1–Orai1 association was likewise detected using the split-Venus assay (Fig. 2g). Consistent with this, treatment with the Ca²⁺ ionophore A23187 triggered robust blebbing in the absence of external stimuli (Supplementary Fig. 4a and Supplementary Movie5).

Together, these findings demonstrate that chick PGCs harbor the full complement of functional SOCE machinery that mediates extracellular Ca²⁺ influx to initiate and sustain bleb formation. Moreover, Ca²⁺ elevation alone is sufficient to induce blebbing in vitro, providing a mechanistic framework for analyzing extravasation in vivo and raising the question of whether the requirement for SOCE is universal or whether distinct Ca²⁺ supply routes have evolved in different lineages.

SOCE-driven bleb formation supports PGC locomotion in vitro
To test whether membrane blebbing contributes to locomotion, we developed an in vitro assay in which chick PGCs actively migrate. We examined two candidate ligands, stromal-derived factor-1 (SDF-1) and stem cell factor (SCF), both implicated in germ cell migration29-31. Chick PGCs express the corresponding receptors, CXCR4 and KIT31,32.

When cultured alone or with COS7 cells expressing either control constructs or SDF-1, PGCs exhibited minimal motility or protrusive activity (Supplementary Fig. 5a-f). By contrast, co-culture with SCF-expressing COS7 cells robustly stimulated motility in more than half of the PGCs, although no directional migration bias was detected (Supplementary Fig. 5a-f). This system therefore provided a tractable assay to interrogate the role of SOCE in PGC migration.

Under SCF stimulation, migrating PGCs generated and maintained large balloon-like protrusions at the leading edge. Live imaging of Lifeact-mCherry and GCaMP6s-expressing cells revealed that these protrusions were actin-poor and Ca²⁺-rich (Fig. 2h,i and Supplementary Fig. 5g,h), consistent with persistent membrane blebs. For comparison, zebrafish PGCs form transient actin-brush structures during blebbing cycles33, whereas chick PGCs lacked such reinforcement. Fluorescently tagged STIM1 and Orai1 co-localized at the leading edge of migrating PGCs, indicating localized SOCE activity (Fig. 2j and Supplementary Fig. 5i). Disruption of SOCE with SKF abolished bleb formation and significantly reduced migration distance (Fig. 2k and Supplementary Fig. 5j). Together, these results demonstrate that SOCE is required not only for bleb formation but also for efficient locomotion of chick PGCs in vitro.

Dominant-negative Orai1 mutant cell-autonomously blocks SOCE in PGCs
To genetically dissect SOCE function in vivo, we established PGCs with inducible, cell-autonomous inhibition of this pathway. We used a Tet-on system34 to drive expression of the dominant-negative Orai1 mutant (E106Q)11, which abolishes Ca²⁺ conduction through Orai1 channels upon Doxycycline (Dox) administration.

In the UAC assay, Orai1 E106Q+ PGCs failed to generate blebs, phenocopying the effect of pharmacological SOCE inhibition (Supplementary Fig. 6a,b). In the SCF-induced migration assay, these cells displayed severely impaired motility relative to controls (Supplementary Fig. 6c,d). Together, these results establish Orai1 E106Q as a robust genetic tool for cell-autonomous suppression of SOCE in chick PGCs, providing a basis for functional analysis in vivo.

SOCE drives bleb-based extravasation and subsequent tissue migration of chick PGCs in vivo
To test whether SOCE-mediated bleb formation is required for PGC extravasation in vivo, we co-infused equal numbers of EGFP-labeled Orai1 E106Q+ PGCs and mCherry-labeled control PGCs into the vasculature of HH15 chick embryos (Supplementary Fig. 7a). Both populations efficiently arrested within the Ex-VaP, demonstrating that SOCE is dispensable for intravascular arrest (Supplementary Fig. 7b,c).

Live imaging 1 hour post-transplantation (hpt) revealed a marked defect in subsequent behaviors: Orai1 E106Q+ PGCs failed to initiate blebbing, remained spherical, and displayed neither intravascular crawling nor TEM (Fig. 3a–c and Supplementary Fig. 7d and Supplementary Movie6). To quantify extravasation efficiency, we transplanted the same cell combinations into HH15 quail embryos and stained for the endothelial marker QH1 at 6 hpt. Quail were used for this analysis because QH1 provides clear and specific endothelial labeling5,35, while comparable antibodies are not available for chick, ensuring acute discrimination between intra- and extravascular PGCs9. Orai1 E106Q+ PGCs extravasated at ~40% lower efficiency than co-infused control PGCs (Fig. 3d,e). By contrast, co-transplanted EGFP+ and mCherry+ controls showed comparable efficiencies, excluding labeling or tracking artifacts (Fig. 3d,e).

We next examined whether SOCE is also required for post-extravasation migration. At E4.5 (2 days post-transplantation), many control PGCs had reached the dorsal mesentery or gonadal primordium, whereas significantly fewer Orai1 E106Q+ PGCs were present in these regions (Supplementary Fig. 7e,f). The relative abundance of mutant cells decreased from 63% in the mesentery to 22% in the gonad, consistent with a progressive defect in tissue migration.

Importantly, in vitro assays confirmed that SOCE inhibition did not affect PGC survival or proliferation (Supplementary Fig. 7g,h). Together, these results establish that SOCE is essential for bleb initiation, vascular exit, and subsequent migration toward the gonad, underscoring its central role in the in vivo trajectory of chick PGCs.

SOCE drives bleb-based transendothelial migration of HT-1080 human cancer cells
Given that SOCE governs membrane blebbing and extravasation in chick PGCs, we asked whether the same mechanism also operates in human cancer cells. Aggressive cancer cells such as HT-1080 fibrosarcoma cells migrate and invade to form metastatic tumors, making them a relevant model for testing whether bleb-based extravasation contributes to malignant dissemination36. When transplanted into the vasculature of HH15 avian embryos, HT-1080 cells rapidly arrested in the YSV within 20 minutes, without evidence of intravascular crawling, and initiated TEM by extending thin pseudopodia, forming large bleb-like protrusions, or combining both modes into mixed-type leading edges (Fig. 4a and Supplementary Fig. 8a,b). These observations are consistent with previous reports indicating that ~10% of extravasating cancer cells employ bleb-driven mechanisms9.

To determine whether these protrusions are bona fide blebs, we imaged HT-1080 cells expressing GCaMP6s and Lifeact-mCherry during TEM. Balloon-like protrusions exhibited localized cytoplasmic Ca²⁺ surges and depletion of cortical F-actin (Fig. 4a–c), recapitulating the molecular hallmarks of blebs observed in chick PGCs.

We next assessed the requirement for SOCE. Inhibition of Arp2/3-mediated actin polymerization with CK-666 induces spherical bleb-like protrusions37, which were abolished by the SOCE inhibitor SKF (Fig. 4d,e and Supplementary Movie7), indicating that SOCE activity is necessary for bleb formation under these conditions. Consistently, thapsigargin treatment also induced robust blebbing in 2D culture (Supplementary Fig. 8c,d), providing additional support for SOCE involvement.

During TEM, GFP-STIM1 accumulated at the plasma membrane within bleb-like protrusions (Fig. 4f), consistent with ER–PM contact formation and localized SOCE activation. To functionally test the role of SOCE in vivo, we generated HT-1080 cells stably expressing Orai1 E106Q. Upon vascular transplantation, Orai1 E106Q+ cells displayed normal arrest and protrusion formation (Supplementary Fig. 8e-h), but showed a significantly reduced rate of TEM compared to controls (Fig. 4g,h and Supplementary Fig. 8i). Notably, actin-rich protrusions such as filopodia were largely preserved under these conditions (Supplementary Fig. 8g,h), indicating that Orai1-dependent Ca²⁺ entry is dispensable for filopodial protrusion formation. Consistent with our previous in vivo analyses showing that HT-1080 cells deploy both bleb-based and actin-rich protrusions during extravasation9, these results suggest that SOCE inhibition preferentially compromises the bleb-based route while leaving actin-rich protrusive modes available.

Importantly, SOCE inhibition did not measurably affect HT-1080 cell viability under the corresponding in vitro conditions (Supplementary Fig. 8j), arguing against nonspecific cytotoxic effects. This provides a mechanistic explanation for the maintained vascular arrest and the incomplete suppression of extravasation. Finally, treatment with the Ca²⁺ ionophore A23187 was sufficient to induce robust blebbing in HT-1080 cells in 2D culture (Supplementary Fig. 4b and Supplementary Movie8). Together, these results establish that SOCE-dependent Ca²⁺ entry from the extracellular space is indispensable for bleb-driven extravasation in HT-1080 cells. This parallels the mechanism observed in chick PGCs and highlights SOCE as a conserved driver of bleb-based TEM across both development and cancer.

IP₃R-mediated Ca²⁺ release drives SOCE-independent bleb formation and extravasation in PC-3 and MDA-MB-231 cancer cells
While SOCE-dependent bleb formation was essential for TEM in both chick PGCs and HT-1080 cells, it remained unclear whether this mechanism was conserved in other metastatic cancer types. To address this, we analyzed PC-3 (prostate cancer) and MDA-MB-231 (breast cancer) cells, which also form bleb-like protrusions during extravasation in the avian model7. Similar protrusions have been reported under culture conditions38,39.

Consistent with bona fide blebs, these balloon-like protrusions were depleted of cortical actin (Fig. 5a). Because GCaMP6s expression impaired viability in these epithelial cancer cells, we instead used chemical indicators to probe Ca²⁺ dynamics. In 2D culture, spontaneously blebbing cells exhibited higher Ca²⁺ signals within blebs compared to the adjacent cell body, as revealed by Fluo-4 AM imaging (Fig. 5b,c; whole-cell view in Supplementary Fig. 9a).

To test whether Ca²⁺ elevation per se could drive blebbing, we applied the ionophore A23187. As in PGCs and HT-1080 cells, A23187 was sufficient to robustly induce bleb formation in both PC-3 and MDA-MB-231 cells (Supplementary Fig. 4c,d and Supplementary Movie9,10), confirming that Ca²⁺ is a conserved driver of bleb initiation. However, manipulation that abolished SOCE-dependent blebbing in PGCs and HT-1080 cells failed to affect PC-3 and MDA-MB-231 cells. Pharmacological SOCE inhibition with SKF had no effect on bleb formation (Fig. 5d-f and Supplementary Movie11,12). Depletion of ER Ca²⁺ stores with thapsigargin, which normally activates SOCE, likewise failed to induce blebbing (Fig. 5g,h). Expression of dominant-negative Orai1 E106Q also left TEM efficiency unchanged in both PC-3 and MDA-MB-231 cells, in stark contrast to the impairment seen in HT-1080 cells (Fig. 5i-k). These results demonstrate that PC-3 and MDA-MB-231 cells do not rely on SOCE. Importantly, SOCE inhibition did not affect the viability of PC-3 or MDA-MB-231 cells during the assays (Supplementary Fig. 9b). Notably, the persistence of blebbing under extracellular Ca²⁺ chelation with BAPTA suggests the involvement of an alternative, intracellular Ca²⁺ source (Fig. 5l-n, Supplementary Movie13,14).

To directly test this possibility, we examined the spatial relationship between ER and Ca²⁺ dynamics. Strikingly, dual visualization with Fluo-4 and ER-ID revealed that ER tubules extend into blebs, and localized Ca²⁺ elevations consistently arose in close proximity to these ER structures (Fig. 6a and Supplementary Fig. 10a). Notably, the morphology of ER extensions within blebs differed between cell types: in MDA-MB-231 cells, a single thicker ER structure was often observed within individual blebs (Fig. 6b), whereas in PC-3 cells multiple thin ER tubules frequently invaded the bleb (Supplementary Fig. 10b). In SOCE-dependent cells, including chick PGCs and HT-1080 cells, ER invasion patterns more closely resembled those seen in PC-3 cells, with multiple thin ER extensions entering blebs (Fig. 2d, Supplementary Fig. 10b). Importantly, these ER extensions did not appear to reach or stabilize at the PM, in contrast to the ER-PM junctions characteristic of SOCE-dependent blebs (Fig. 6b and Supplementary Fig. 10b). This indicates that, rather than engaging in SOCE, these epithelial cancer cells mobilize ER-derived Ca²⁺ directly within blebs to fuel their expansion.

Consistent with this model, pharmacological inhibition of IP₃Rs, a major class of ER Ca²⁺ release channels, with 2-APB, as well as the more specific inhibitor Xestospongin C, markedly reduced blebbing (Fig. 6c,d and Supplementary Fig. 10c-f and Supplementary Movie15,16). To complement these pharmacological observations and to further strengthen pathway specificity, we next examined the effect of genetic depletion of IP₃R3, the predominant isoform in both cell lines(Supplementary information 2)40. To minimize potential off-target effects, two independent siRNAs targeting distinct regions of ITPR3 were used (Supplementary Fig. 10g), both of which yielded consistent phenotypic outcomes. IP₃R3 knockdown significantly reduced the frequency of blebs and other protrusions in vitro (Fig. 6e,f and Supplementary Fig. 10h), without affecting overall cell viability (Supplementary Fig. 10n). In vivo, IP₃R3-deficient cells exhibited normal vascular arrest but markedly reduced extravasation efficiency (Fig. 6g,h and Supplementary Fig. 10i-m), implicating IP₃R3 in promoting TEM through its control of Ca²⁺-dependent protrusive activity.

Together, these findings define a distinct, SOCE-independent mechanism in which IP₃R3-mediated Ca²⁺ release from ER reservoirs drives extravasation in PC-3 and MDA-MB-231 cells. This establishes a lineage-specific divergence of Ca²⁺ supply routes: SOCE operates in chick PGCs and HT-1080 cells, whereas IP₃R3-mediated release predominates in PC-3 and MDA-MB-231 cells (Fig. 7).

Importantly, this divergence in Ca²⁺ supply mode does not appear to be simply explained by differential expression levels of core Ca²⁺ signaling components such as STIM, Orai, or IP₃Rs. Analysis of publicly available transcriptomic datasets indicates that these molecules are expressed at comparable levels across HT-1080, PC-3, and MDA-MB-231 cells. This suggests that the choice of Ca²⁺ supply route—SOCE versus IP₃R-mediated ER release—is likely determined by functional deployment or contextual regulation of Ca²⁺ signaling pathways, rather than by their absolute expression levels (Supplementary Table 2).

Methods
Animals, staging, and animal care
Fertilized chicken (Gallus gallus domesticus, White leghorn) eggs and fertilized quail (Coturnix japonica) eggs were purchased from Yamagishi poultry farm (Mie, Japan) and from Nagoya University through the National Bio-Resource Project of the MEXT, Japan, respectively. Eggs were incubated at 38.5℃, and embryos were staged by Hamburger and Hamilton’s stage16. All animal experiments were performed with the approval of the Institutional Animal Care and Use Committees at Kyushu University.

Plasmid constructions
pT2A-BI-Gap-TdTomato-TRE: pT2AL200R150G vector55 was digested with XhoI-BglII. These sites were blunt-ended, and inserted with the fragment of pBI-TRE (Clontech) containing the bidirectional tetracycline-responsive element (TRE) with two minimal promoters of CMV in both directions, and two polyA-additional sequences of the rabbit beta globin gene. This vector was designated as pT2A-BI-TRE. The full-length of Gap-TdTomato56 was amplified by PCR, and subcloned into the SpeI-HindIII site of pT2A-BI-TRE. pT2A-BI-Gap-TdTomato-TRE- (SCF or SDF-1): The ORF of chicken SCF or chicken SDF-157 was subcloned into the MluI-EcoRV site of pT2A-BI-Gap-TdTomato-TRE. The ORF of SCF was amplified and isolated from cDNA derived from E2.5 whole chick embryo by PCR. pT2A-BItight-EGFP-TRE: pT2AL200R150G vector was digested with XhoI-BglII. These sites were blunt-ended, and inserted with the fragment of pBItight-TRE (Clontech) containing the improved bidirectional tetracycline-responsive element (TRE) with two minimal promoters of CMV in both directions, and two polyA-additional sequences of the rabbit beta globin gene. This vector was designated as pT2A-BItight-TRE. The full-length of EGFP was amplified by PCR, and subcloned into the EcoRI-PstI site of pT2A-BItight-TRE. pT2A-BItight-EGFP-TRE-Orai1 E106Q: The ORF of human Orai1 with E106Q mutation and myc tag (addgene #22754) was amplified by PCR, and subcloned into the MluI-EcoRV site of pT2A-BItight-EGFP-TRE. pT2A-BItight-mCherry-TRE: The full-length of mCherry was amplified by PCR, and subcloned into the EcoRI-BglII site of pT2A-BItight-TRE. pT2A-BItight-GCaMP6s-2AP-mCherry-TRE: The consecutive sequences of GCaMP6s-2AP-mCherry58 was amplified by PCR, and subcloned into the EcoRI site of pT2A-BItight-TRE by In-Fusion®︎ HD cloning kit (TaKaRa). pT2A-BItight-Lifeact-mCherry-TRE: Lifeact-mCherry was amplified by PCR using pT2A-BI-TRE-LAP2bcGFP-Lifeact-mCherry9 as a template, and subcloned into the MluI-EcoRV site of pT2A-BItight-TRE. pT2A-CAGGS-EGFP-CAAX-IRES2-PuroR: The sequences of IRES2 and PuroR were amplified by PCR, and subcloned into the XhoI-EcoRI site and EcoRI-BglII site into pT2A-CAGGS, respectively. This vector was designated as pT2A-CAGGS-IRES2-PuroR. The full-length of EGFP-CAAX was amplified by PCR, and subcloned into the PstI-XhoI site of pT2A-CAGGS-IRES2-PuroR. pT2A-CAGGS-MRLC1-EGFP-IRES-PuroR: MRLC1-EGFP was amplified by PCR, and subcloned into the NotI-XhoI site of pT2A-CAGGS-IRES2-PuroR. pT2A-CAGGS-Tet3G-IRES2-PuroR: The full-length of Tet3G was amplified by PCR, and subcloned into the NotI-XhoI site of pT2A-CAGGS-IRES2-PuroR. pT2A-BItight- mCherry-CAAX-TRE: mCherry-CAAX was amplified by PCR, and subcloned into the BglⅡ site and EcoRV site of pT2A-BItight-TRE. pT2A-BItight-stim1-EGFP-TRE: Stim1-EGFP was amplified by PCR (addgene #210370), and subcloned into the MluI-EcoRV site of pT2A-BItight-TRE.

Primer sequences are as described below.
Gap43-SpeI-F: agaactagtggatccccgcggATGTTGTGCTGTATGAGAAGAACCAAGC
TdTomato-HindIII-R: gacaagcttgaattcTTACTTGTACAGCTCGTCCATGCCGTACAG
SDF-1-MluI-F: cagacgcgtATGGACCTCCGCGCCCTGGCTCTGSCTCGCCT
SDF-1-EcoRV-R: agagatatcTTACTTGTTTAAAGCTTTCTCSCAGATATTCC
SCF-MluI-F: aattacgcgtATGAAGAAGGCACAAACTTGGATTATCAC
SCF-EcoRV-R: aattgatatCTACACTTGTAGATGTTCTTTTTCTTTTTGCTGCAACA
EGFP-EcoRI-F: ggagaattcATGGTGAGCAAGGGCGAGGAGCTGTTCACCG
EGFP-PstI-R: agactgcagTTACTTGTACAGCTCGTCCATGCCGAGAGT
Orai1 E106Q-MluI-F: ctgacgcgtACCATGCATCCGGAGCCCGCCCCGCCCC
Orai1 E106Q-EcoRV-R: ggagatatcCTAGEAGATCTTCCTCAGAAATGAGC
mCherry-EcoRI-F: ggagaattcATGGTGAGCAAGGGCGAGGAGGATAACATGG
mCherry-BglII-R: caggagatctTTACTTGTACAGCTCGTCCATGCCGCCGGT
GCaMP6s-inf-F: gtcagatcgccTtggaATGGGTTCTCATCATCATCATCATCATGGTATGG
mCherry-inf-R: gactgcagcctcaggagatcTTACTTGTACAGCTCGTCCATGCCGCCGG
Gap43-MluI-F: attacgcgtcggcaccATGCTGTGCTGTATGAGAAGAACCAAGCAGGTG
mOrange-EcoRV-R: tctgataTCACTTGTACAGCTCGTCCATGCCGCCG
stim1-EGFP-MluI: cgatacgcgtATGGATGTATGCGTCCGTCT
stim1-EGFP-EcoRV: tggagatatcTTACTTGTACAGCTCGTCCA
IRES2-XhoI-F: aatctcgagGCCCCTCTCCCTCCCCCCCCCCTAAC
IRES2-EcoRI-R: aatgaattcTGTGGCCATATTATCATCGTGTTTTTCA
PuroR-EcoRI-F: agagaattcATGACCGAGTACAAGCCCACGGTGCGCCTCG
PuroR-BglII-R: aaaagatctTCAGGCACCGGGCTTGCGGGTCATGCACCAGGT
EGFP-PstI-F: aagctgcagcggcaccATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGG
CAAX-XhoI-R: gcctcgagTTACATAATTACACACTTTGTCTTTGACTTCTTTTTCTTCT
MRLC1-NotI-F: tgcagcggccgcATGTCCAGCAAGCGGGCCAA
EGFP-XhoI-R: gggcctcgagTTACTTGTACAGCTCGTCCATG
Tet3G-NotI-F: gcagcggccgcATGTCTAGACTGGACAAGAGCAAAGTC
Tet3G-XhoI-R: ggcctcgagTTACCCGGGGAGCATGTCAAGGTCAAAAT
mCherry-EcoRI-F: ggagaattctagatatcggcaccATGGTGAGCAAGGGCGAGGAGGATAA
CAAX-BglII-R: aggagatcTTTACATAATTACACACTTTGTCTTTGACTTCTTTTTCTTC

Establishment and maintenance of PGCs in culture
Circulating PGCs along with blood cells were harvested from blood of HH 15 chicken embryos, and were cultured in Ca²⁺-free DMEM (Gibco) diluted with water, containing FGF2 (Wako), Activin A (APRO Science), and chicken serum (FAcs medium) according to the method previously described 59,60. After one month, expanded PGCs were cryo-preserved at -80℃ in Bambanker (NIPPON Genetics) until used for experiments.

Maintenance of HT-1080, PC-3, and MDA-MB-231 in culture
HT-1080 cells (JCRB Cell Bank, JCRB9113) and MDA-MB-231 cells (JCRB Cell Bank, JCRB1559) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Nakarai tesque) containing 4.5g/l Glucose, L-Glutamine, and sodium pyruvate, supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco), GlutaMaxTM (Gibco), and 1% (v/v) penicillin-streptomycin-amphotericin B (PSA; FUJIFILM Wako Pure Chemical Corporation).
PC-3 cells (ATCC, CRL-1435) were cultured in Roswell Park Memorial Institute medium 1640 (RPMI1640; Wako) supplemented with 10% (v/v) FBS (Gibco), GlutaMaxTM (Gibco), and 1% (v/v) PSA.
All cells were incubated at 37°C in a humidified atmosphere of 5% CO2 and passaged using 0.05% trypsin-EDTA (Gibco).

Plasmid transfection and establishment of gene-manipulated PGC lines and cultured cell lines (COS7, HT-1080, PC-3, MDA-MB-231)
PGCs: Cultured PGCs (5 x 104) were washed with OPTI-MEM (Gibco) and seeded into 96-well plates containing 100 μl of FAot medium60 (supplemented with ovo-transferrin instead of chicken serum) without heparin and PSA for 3 h. A transfection mixture containing 0.2 μg of total plasmid DNA and 0.5 μl of Lipofectamine 2000 (ThermoFisher Sciencetific) in 50 μl of OPTI-MEM was then added. After 6 h, the medium was replaced with conventional FAcs medium containing heparin and antibiotics.
Seven plasmid sets were used for PGC transfection:
1. pT2A-BItight-GCaMP6s-2AP-mCherry + pT2A-CAGGS-Tet3G-IRES2-PuroR + pCAGGS-T2TP61
2. pT2A-BItight-Lifeact-mCherry-TRE + pT2A-CAGGS-Tet3G-2AP-PuroR4 + pCAGGS-T2TP
3. pT2A-BItight-EGFP-TRE + pT2A-CAGGS-Tet3G-IRES2-PuroR + pCAGGS-T2TP
4. pT2A-BItight-mCherry-TRE + pT2A-CAGGS-Tet3G-IRES2-PuroR + pCAGGS-T2TP
5. pT2A-BItight-EGFP-TRE-Orai1 E106Q + pT2A-CAGGS-Tet3G-IRES2-PuroR + pCAGGS-T2TP
6. pT2A-BItight-stim1-EGFP-TRE + pT2A-BItight-mCherry-CAAX-TRE + pT2A-CAGGS-Tet3G-IRES2-PuroR + pCAGGS-T2TP
7. pT2A-CAGGS-MRLC1-EGFP-IRES-PuroR + pCAGGS-T2TP
Following transfection, PGCs were cultured in FAcs medium supplemented with 0.1-0.3 μg/ml puromycin for 3 days. In addition, plasmids encoding Orai1-mCherry (pCAGGS-neo vector) and EGFP-tagged STIM1 (pCAGGS-neo vector)11, as well as BiFC constructs pcDNA3.1-STIM1–Venus173C (Addgene: #87619) and pcDNA3–Venus173N–ORAI1 (Addgene: #87618), were introduced using the same method. Cells were used for experiments one day after transfection.
COS7 cells: COS7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented 1.5g/L sodium bicarbonate, 10% fetal bovine serum (FBS), 50 IU/ml penicillin and 50mg/ml streptomycin at 37℃. Cells were transfected with following sets using Lipofectamine 2000 according to the manufacture’s instruction:
1. pT2A-BI-GapTdTomato-TRE + pT2K-M2-IRES2-NeoR57 + pCAGGS-T2TP
2. pT2A-BI-GapTdTomato-TRE-SCF + pT2K-M2-IRES2-NeoR + pCAGGS-T2TP
3. pT2A-BI-GapTdTomato-TRE-SDF-1 + pT2K-M2-IRES2-NeoR + pCAGGS-T2TP
Stable lines were selected in G418-containing medium.
HT-1080 cells (lipofection): HT-1080 cells (1 × 106) were washed with OPTI-MEM and seeded into 6-well plates (1 ml of medium without PSA) for 18-24 h (overnight). A transfection mixture (4.0 μg of total plasmid DNA + 7.0 μl of Lipofectamine 2000 in 250 μl of OPTI-MEM) was added. Two plasmid sets were used:
1. pT2A-BItight-Lifeact-mCherry-TRE + pT2A-CAGGS-Tet3G-2AP-PuroR + pCAGGS-T2TP
2. pT2A-BItight-EGFP-TRE + pT2A-CAGGS-Tet3G-2A-PuroR + pCAGGS-T2TP
12 hours later, the medium was replaced with conventional medium containing antibiotics. After 2 days, cells were cultured in 2-5 μg/ml puromycin for 2 days. EGFP-positive cells were then sorted using an SH800 cell sorter (Sony).
HT-1080 cells (electroporation): HT-1080 cells (1 × 106) were also electroporated using the Neon Transfection System (Invitrogen). For electroporation, 6 × 104 cells were transfected with:
1. pT2A-BItight-stim1-EGFP-TRE + pT2A-BItight-mCherry-CAAX-TRE + pT2A-CAGGS-Tet3G-IRES2-PuroR + pCAGGS-T2TP
2. pT2A-BItight-GCaMP6s-2AP-mCherry + pT2A-CAGGS-Tet3G-IRES2-PuroR + pCAGGS-T2TP
Cells were cultured as above with antibiotic replacement at 12 h and puromycin selection after 2 days.
PC-3 and MDA-MB-231 cells: PC-3 or MDA-MB-231 cells (1× 106) were seeded into 6-well plates (1 ml of medium without PSA) for 18-24 h. A transfection mixture (4.0 μg of total plasmid DNA and 7.0-10 μl of Lipofectamine 2000 in 250 μl of OPTI-MEM) was then added. Four plasmid set was used for transfection:
1. pT2A-BItight-Lifeact-mCherry-TRE + pT2A-CAGGS-Tet3G-2AP-PuroR + pCAGGS-T2TP.
2. pT2A-BItight-Lifeact-mCherry-TRE + pT2A-CAGGS-Tet3G-2ASP-PuroR
3. pT2A-BItight-EGFP-TRE-Orai1 E106Q + pT2A-CAGGS-Tet3G-2A-PuroR
4. pT2A-BItight-EGFP-TRE + pT2A-CAGGS-Tet3G-2A-PuroR.
For set 1, the medium was replaced with conventional medium containing antibiotics 12 h after transfection. After 2 days, cells were further cultured in medium containing 2-5 μg/ml puromycin for 2 days. For sets 2-4, the medium was replaced with conventional medium containing antibiotics 12 h after transfection, and cells were used for experiments one day later without puromycin selection.

in vitro under-agarose compression assay (UAC assay)
This method was modified from the protocol reported by Heit et al.20. To control adhesion of PGCs with dish bottom, the surface of glass bottom dish (Eppendorf) was treated by 0.5 g/L of Pluronic®F-127 (Sigma-Aldrich) for 30 min. The treated dishes was dried by air for 15 min before use.
2 x Hank’s balanced salt solution (Gibco) and FAcs medium (1mM CaCl2) were mixed in 1:2 and warmed up to 70℃. UltraPureTM Agarose (Invitrogen) solution (48 mg/ml) was prepared by microwave. HBSS/FAcs solution and UltraPureTM Agarose solution were mixed in 1.99:1.7. 3 ml of mixed solution poured onto the glass bottom and solidified at room temperature (RT). 250 μl of FAcs was added to the gel and placed at the condition of 37℃, 5% CO2 for 30 min for equilibration. In case of chemical addition, BAPTA (Toronto Research Chemicals Inc), 2-APB (R&D Systems), SKF96365 (Wako), BTP2 (MERCK) was added to the mixed solution of HBSS/FAcs and UltraPureTM Agarose and each final concentration is 100 μM, 100 μM, 50 μM, 1μM, respectively.
The gel was removed from the glass bottom dish. After removing the remaining solution on the dish, 2.5×105 PGCs and 5.0×105 FluoSpheresTM Polystyrene Microspheres (Invitrogen, F8836) with a diameter of 10 μm were suspended in 5 μl of FAcs medium and seeded on the glass bottom dish and then the gel was put back on. 50 μl of FAcs and Doxycycline (Dox; 1 g/ml, final concentration; Clontech) were added on the gel. At this time, we adjusted the final concentration of Dox to 1 g/ml.

in vitro migration assay
PGCs (1.4×104 cells/μl) and COS7 cells (2.8×106 cells/μl) were suspended in 4-5% and 6-7% Matrigel® Basement Membrane Matrix Growth Factor Reduced, Phenol Red Free (Corning), respectively, prepared in /FAcs medium containing 1mM CaCl2. The suspensions were seeded adjacent to each other in glass bottom dishes (Matsunami). The dishes were incubated at 37℃ for 20 min to allow Matrigel solidification, after which FAcs medium (1mM CaCl2) containing Dox (1 g/ml, final concentration) was added on top of the Matrigel. Where indicated, inhibitors were added to FAcs medium at the following final concentrations: BAPTA (100 μM), 2-APB (100 μM), SKF96365 (50 μM).

2D culture of cancer cells with chemical inhibitors and Ca²⁺ ionophore
HT-1080, PC-3, and MDA-MB-231 cells (1.8 × 103 cells/μl) were seeded onto the glass-bottom dishes (Matsunami). After incubation at 37 °C for 15 h, live imaging was initiated. The following chemicals were then added to the culture medium at their final concentrations: CK-666 (120 μM), SKF96365 (50 μM), Thapsigargin (1 μM), BAPTA (100 μM), 2-APB (75 μM), A23187 (50 μM) and Xestospongin C (5 μM, Cayman Chemical).

3D culture of PGCs with thapsigargin or A23187 treatment
PGCs (1.4×104 cells/μl) were suspended in 4-5% Matrigel, prepared by diluting in FAcs medium containing 1mM CaCl2, and seeded into glass bottom dishes (Matsunami). The dish was incubated at 37℃ for 20 min to allow Matrigel solidification. FAcs medium (1mM CaCl2) containing Dox (1 g/ml, final concentration) was then overlaid onto the Matrigel. Thapsigargin (2 μM, final concentration) or A23187 (10 μM, final concentration) was added to the overlaid FAcs medium.

PGC transplantation into embryo
For back-infusion, cultured PGCs were collected by centrifugation at 63×g for 5 minutes, washed several times in OPTI-MEM, and resuspended at 5,000 cells/l. A 1 l suspension was injected into the heart of HH15 chick or quail embryo using a fine glass capillary. To visualize blood vessels, Alexa Fluor™ 594- or Alexa Fluor™ 488-conjugated acetylated low-density lipoprotein (594-AcLDL or 488-AcLDL; ThermoFisher Sciencetific, L35353 and L23380) was diluted 1:2 in OPTI-MEM and co-injected with PGCs. For the tet-on induction, 1 g/ml Dox was added to PGC cultures in FAcs medium 24 hours before transplantation. For in ovo induction, 0.5 ml of 100 μg/ml Dox solution was injected between the embryo and yolk as described previously34. Embryos were incubated at 38.5℃ until intended developmental stage.

Transplantation of HT-1080, PC-3, and MDA-MB-231 cells into embryo
HT-1080, PC-3, and MDA-MB-231 cells were detached from culture dishes using 0.05% trypsin-EDTA in phosphate-buffered saline (PBS) and trypsinization was quenched with an equal volume of FBS. Cells were collected by centrifugation at 63×g for 5 min, washed several times with OPTI-MEM, and resuspended at 25,000 cells/l. A 1 l suspension was injected into the heart of HH15 chick or quail embryos using a fine glass capillary. To visualize blood vessels, either 12.5% Rhodamine-Lens culinaris lectin (Rho-LCA; Vector Laboratories, L-1040) or 12.5% FITC-conjugated Lens culinaris agglutinin (FITC-LCA; Vector Laboratories, FL-1041) diluted in OPTI-MEM was co-injected with cells. Embryos were incubated at 38.5 ℃ until the intended developmental stage.

Fixation, whole-mount, and section immunostaining
Whole-mount immunostaining: Embryos containing infused PGCs were fixed in 4% PFA in PBS at 4 ℃ for 20 h. For immunostainings with QH1, EGFP, and mCherry in whole quail embryos, samples were washed 3 times for 60 minutes each in TNTT buffer (0.1 M Tris-HCl, pH 7.5; 0.15 M NaCl; 0.05% Tween 20; 0.5% TritonX-100) at 4 ℃. To inactivate endogenous peroxidase, embryos were immersed in 3% hydrogen peroxide in methanol for 5 days at 4 ℃, followed by 6 washes in TNTT (60 min each, 4 ℃). Blocking was performed in 1% Blocking Reagents (Roche) in TNTT for 1 hour at 4 ℃. The blocked samples were treated 4 days at 4℃ with primary antibodies diluted in blocking solution: QH1 (Hybridoma Bank, AB_531829, 1:200), anti-EGFP goat polyclonal antibody (GeneTex, GTX266673, 1:1,000), and anti-mRFP rabbit polyclonal antibody (ROCKLAND, 600-401-379, 1:1,000). After 6 washes in TNTT (60 min each, RT), samples were incubated for 4 days at 4 ℃ with secondary antibodies: Alexa Fluor 555-conjugated donkey anti-rabbit IgG (Invitrogen A31572, 1:500), HRP-linked sheep anti-mouse IgG (Cytiva NA931V, 1:500), and Alexa Fluor 488-conjugated donkey anti-goat IgG (Invitrogen A11055, 1:500) in 1% blocking solution. Finally, embryos were washed 3 times for 1 h each in TNTT (RT). Fluorescence signal amplification was performed by incubating embryos in 1% Cyanine 5 reagent (PerkinElmer FP1171) with amplification diluent (PerkinElmer FP1135) for 15 min at RT, followed by 3 washes in TNTT (1 h each, RT). Embryos were cleared by sequential immersion in 40% glycerol/PBS (1 hour, 4 ℃), 60% glycerol/PBS (2-3 hours, 4 ℃), and 80% glycerol/DDW overnight at 4℃.
Section immunostaining: For section staining, fixed embryos were washed twice with PBS, and cryoprotected in 30% sucrose/PBS at 4℃ for 3-5 hrs. Samples were then incubated overnight at 4 ℃ in a 1:2 mixture of 30% sucrose/PBS and O.C.T. Compound (Sakura Finetek Japan), followed by embedding in 100% O.C.T. and freezing at -30℃. Cryosections (12 µm) were washed 3 times in TNTT and blocked in 1% Blocking Reagents/TNTT for 1 h at RT. Sections were incubated at 4℃ with primary antibodies: anti-EGFP goat polyclonal antibody (GeneTex, GTX266673, 1:1,000) and anti-mRFP rabbit polyclonal antibody (ROCKLAND, 600-401-379, 1:1,000) diluted in blocking solution. After 3 washes in TNTT (5 min each, RT), sections were incubated for 1 h at RT with secondary antibodies: Alexa Fluor 555-conjugated donkey anti-rabbit IgG (Invitrogen A31572, 1:500) and Alexa Fluor 488-conjugated donkey anti-goat IgG (Invitrogen A11055, 1:500) in 1% blocking solution. Sections were washed 3 times in TNTT and mounted in Fluoromount-GTM with DAPI (Invitrogen).

Measurement of cell viability and functional validation of Ca²⁺ signaling perturbations
PGCs and human cancer cell lines (HT-1080, PC-3, and MDA-MB-231) were cultured in their respective media. For cell viability assays, culture media were replaced with Opti-MEM prior to cell collection. Cells were harvested according to cell type and experimental condition. PGCs were collected directly, whereas HT-1080, PC-3, and MDA-MB-231 cells, which are adherent, were collected by combining floating cells in the culture medium with adherent cells detached by trypsinization. All cell suspensions were centrifuged at 63×g for 5 min.
To genetically inhibit store-operated Ca²⁺ entry (SOCE), PGCs and HT-1080 cells carrying a doxycycline-inducible Orai1 E106Q construct were treated with doxycycline to induce transgene expression. Cell viability was assessed 24 h after doxycycline addition in HT-1080 cells, and at 24, 48, and 72 h after induction in PGCs, as indicated.
For pharmacological and knockdown-based perturbation of Ca²⁺ signaling, PC-3 and MDA-MB-231 cells were seeded at a density of 50,000 cells per well in 24-well plates (Thermo Fisher Scientific) and allowed to adhere for 6 h prior to treatment. Cells were then subjected to SOCE inhibition by treatment with the SOCE inhibitor BTP2 or analyzed following IP3R3 knockdown, as indicated. Twenty-four hours after BTP2 treatment, or at the indicated time points following IP3R3 knockdown, cells were collected as described above.
Total cell numbers and viability were assessed by trypan blue exclusion using a TC20 automated cell counter (Bio-Rad).

Knockdown of IP3R3 in PC3 and MDA-MB-231 cells
PC-3 or MDA-MB-231 cells (1 × 10⁶) were seeded onto 6-well plates with 1 mL of medium without PSA and cultured for 18–24 h. The medium was then replaced with 1 mL of Opti-MEM, and the RNAi transfection mixture (containing 5 μL of DsiRNA [final concentration: 50 nM] and 5 μL of RNAiMAX (ThermoFisher Sciencetific), diluted in 100 μL of Opti-MEM) was added. Cells were incubated for 24 h, washed three times, and subsequently maintained in complete culture medium at 37 °C for an additional 72 h prior to qPCR analysis or other experiments.
DsiRNAs targeting ITPR3 (IP3R3) were purchased from Integrated DNA Technologies (IDT)：
1. ITPR3. 13. 1: GGA CUG ACA AGA AUA ACG UGA U
GC UCG UUA UUC UUG
2. ITPR3. 13. 2: GAC AUC AUG GUC ACU AAG GGU UGG GCU UAG UGA CC

Quantitative PCR analysis of IP3R3 knockdown
Total RNA was extracted from PC-3 and MDA-MB-231 cells 72 h after transfection using the NucleoSpin RNA XS kit (Takara), according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara). Quantitative PCR (qPCR) was performed with SYBR™ Green qPCR Master Mix (Thermo Fisher Scientific) on a CFX Connect Real-Time PCR Detection System (Bio-Rad).
Primer sets used:
Forward: ccctcaatctgaccaacaaga
Reverse: cggtagatgaggtcaaagag

Image acquisition and processing
In ovo live imaging: Cultured embryos was imaged using an MVX10 microscope (Olympus) or a S9 D Stereo Microscope (Leica). Images were acquired with high-speed recording software (Hamamatsu) or Flexacam C3 (Leica). Acquired images were enlarged and processes using Fiji-ImageJ (NIH).
Ex vivo live imaging: Samples on glass-bottom dishes (Matsunami) were placed in an incubation chamber (37 °C, 5% CO2 : TOKAIHIT STX) equipped with an IX83 inverted microscope (Olympus) interfaced with a Dragonfly 200 spinning-disk confocal system (Oxford Instruments). Images were captured with a scientific camera and acquired using Fusion software (Oxford Instruments). Images were enlarged and processed using Imaris (Oxford Instruments) or Fiji-ImageJ (NIH).
In vitro imaging: For live imaging of under-agarose assay and in vitro migration assay, samples on the glass-bottom dishes (Greiner bio-one) were maintained in an incubation chamber (37 °C, 5% CO2 : TOKAIHIT STX) on an IX83 inverted microscope with a Dragonfly 200 spinning-disk confocal system. Images were acquired with a scientific CMOS camera using Fusion software. Z-stacks were processed, enlarged, and reconstructed along the Z-axis using Imaris. ER-ID subtraction images were generated by removing ER-derived Ca2+ signals from Fluo-4 to highlight cytoplasmic Ca2+ dynamics.
Fixed whole-mount imaging: Whole-mount fixed samples were imaged with the Dragonfly 200 spinning-disk confocal system. Images were captured with a scientific camera and acquired using Fusion software. Acquired Z-series images were deconvoluted using Huygens (Scientific Volume Imaging) and processed for 3D reconstruction in Imaris. Iso-surfaces rendering with texture mapping and plane clipping (dorsal-ventral orientation) was performed for visualization.
Ca²⁺ imaging and fluorescence quantification: GCaMP6s fluorescence intensity was quantified and normalized to the fluorescence intensity of co-expressed mCherry in the same region of interest. The values are presented as relative GCaMP6s fluorescence (GCaMP6s/mCherry). GCaMP6s and mCherry were expressed from a single vector in which the two coding sequences were linked by a 2A peptide and driven by the same promoter.
Visualization of the endoplasmic reticulum in PGCs: STIM1-EGFP–expressing PGCs were collected by centrifugation at 63×g for 5 min and resuspended in Assay Buffer from the ER-ID® Red Assay Kit (Enzo). Dual Detection Reagent and Hoechst 33342 (both included in the kit) were added at 2 μl per 1 ml of suspension and incubated for 15 min at RT. The cells were then collected again by centrifugation at 63×g for 5 min, resuspended in FAcs medium, and maintained at 37 °C with 5% CO₂ until imaging.
Visualization of ER, intracellular Ca²⁺, and PM in PC-3 and MDA-MB-231 cells: PC-3 and MDA-MB-231 cells (1.8 × 10³ cells/μl) were seeded onto glass-bottom dishes (Matsunami) and incubated at 37 °C for 15 h. After three washed with PBS, cells were stained with ER-ID Dual Detection Reagent (1:1,000 dilution) for 20 min at RT, followed either by loading with Fluo-4 AM (3 μM in Opti-MEM; ThermoFisher) for 30 min at 37 °C to visualize intracellular Ca²⁺, by staining with CellMask Green (1:1,000 dilution in Opti-MEM; ThermoFisher) for 15 min at 37 °C to visualize the PM. After three additional PBS washes, cells were maintained in complete culture medium at 37 °C until imaging.
Visualization of PGCs expressing STIM1–Venus173C and Venus173N–ORAI1: To visualize fluorescence in PGCs expressing STIM1–Venus173C and Venus173N–ORAI1, cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min at room temperature either after the UAC assay or following thapsigargin treatment. For samples subjected to the UAC assay, the agarose layer was carefully removed prior to cell collection. Cells were then collected and centrifuged at 63×g for 5 min, and the resulting cell pellet was mounted in Fluoromount-G™ with DAPI (Invitrogen) to prepare microscopy slides.
Creating Kymographs of GCaMP6s- or Lifeact-expressing PGCs: Kymographs were generated from time-lapse movies of PGCs expressing GCaMP6s or Lifeact using ImageJ. Line selections were along the cell periphery, and kymographs were constructed to visualize the spatiotemporal dynamic of Ca²⁺ signals or actin remodeling during bleb formation.

Quantification and statistical analysis
PGC quantification: Areas and fluorescence intensities of GCaMP6s and mCherry signals in blebs and cell bodies (in vitro under-agarose assay and in vivo) were measured using ImageJ. Migration tracks and distances in the in vitro migration assay were quantified using the Manual tracking plug-in in imageJ. The number of protrusion-forming PGCs (in vitro migration assay and Ex-VaP) and arrested PGCs (5 h after infusion in Ex-VaP) were counted manually. EGFP+ or mCherry+ PGCs were also counted manually at 6 h after infusion in Ex-VaP and at 2 days after infusion in the dorsal mesentery and gonads.
Cancer cell quantification: Protrusion formation in 2D culture was quantified manually. The number of control or Orai1 E106Q-expressing HT-1080 cells passing through the imaging field in the YSV was manually counted from time-lapse movies. Extravasated cancer cells were quantified by counting EGFP+ cells in the YSV at 3.5 h after infusion. For Ca²⁺ signal quantification, fluorescence intensities were measured in membrane blebs and adjacent cytosolic regions while excluding nuclear areas from the analysis.

Statistics
All statistical analyses were performed using GraphPad Prism (version 11.0.0). Statistical details are indicated in the figure legends and provided in the Source Data file. Box plots in the main figures and Supplementary Figures were generated using GraphPad Prism (version 11.0.0), and the remaining graphs were prepared using Microsoft Excel. All box-and-whisker plots show medians (center lines), interquartile ranges (boxes; 25th–75th percentiles), and minimum to maximum values (whiskers). Bar graphs and line graphs represent mean values. The definitions of error bars are provided in the corresponding figure legends.

RNA-seq data analysis
To assess the expression of STIM1/2 and ORAI1/2 transcripts, we analyzed published RNA-seq data of chicken PGCs (GSE188689). Transcript abundance was quantified as transcripts per million (TPM) using kallisto (v0.46.0) with default settings, pseudoaligning the reads to the chicken reference transcriptome (GCF_016699485.2).
For human cancer cell lines, processed RNA-seq gene expression data (TPM values estimated by RSEM) were obtained from the Cancer Cell Line Encyclopedia (CCLE) via the DepMap portal. We used the CCLE 2019 release and extracted TPM values for indicated genes and cell lines from the file CCLE_RNAseq_rsem_genes_tpm_20180929.txt.gz. As the data were already normalized to TPM by the CCLE pipeline, no additional normalization was applied beyond subsetting the relevant genes and cell lines.

Software version
Image analysis was performed using Fiji (ImageJ, version 2.16.0/1.54p), Imaris (x64, version 9.6.0), Huygens (version 17.10), and Fusion (version 2.2.0.49). Graphs and box plots were generated using GraphPad Prism (version 11.0.0). RNA-seq transcript quantification was performed using kallisto (version 0.46.0).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is an interesting paper that uses the avian embryo system as a model to assess the role of blebbing in extravasation of PGC cells as well as three cancer cell lines. Using elegant imaging in vivo they convincingly show a role for Ca2+ in bleb formation and extravasation. Surprisingly the source of Ca2+ changes based on the cell type, requiring SOCE in PGC and HT-1080 and IP3-dependent Ca2+ release in PC1 and MDA231 cells. The functional data is strong and convincing. However, the imaging data for SOCE effectors in the bleb is weak and needs to be strengthened. More importantly a more detailed time course of the Ca2+ transient, actin polymerization, and bleb formation is critical to better understand the mechanisms involved. Currently it is not clear what is the role of the Ca2+ signal, bleb initiation, expansion, maintenance, etc.\nSpecific comments:\nIt would be helpful to have timelapse of coincident actin formation and Ca2+ signals within the blebs to determine their relative time courses.\nExtended figure 3: the STIM1 and Orai1 staining looks normal, however there is an issue with the ER-ID staining not clear what the large blobs in the ER are? Please explain.\nThe functional data supporting a role for SOCE in blebbing in PGCs is strong but the imaging support is rather weak. More in depth analysis is required to convincingly show that STIM1-Orai1 junction at formed in the blebs. The data in Fig. 2d and 2h are not convincing and actually argue against SOCE activation. The data show ER in the bleb to is close to the plasma membrane consistent with ER-PM contact sites. However, SOCE is typically marked by the formation of STIM1-Orai1 puncta . One would need to fix to stabilize the blebs and then quantify STIM1-Orai1 puncta indicative of SOCE through z-stacks to be comprehensive and compare to the cell body. Also what happens to these puncta under thapsigargin treatment. Are GCAMP measurements in the blebs rapid enough to detect Ca2+ entry through SOCE?\nFor the fluo4 measurements in Fig. 5 show the entire cell as it is not clear what the levels of Ca2+ are across the entire cell compared to the bleb.\n2-APB blocks both SOCE and the IP3R so it is not a good drug to separate the two pathways. Better use a more specific IP3R blocker to support the knockdown experiments. The knockdown data are much stronger and more convincing so consider moving them into the main figures while removing the pharmacology data.", "reviewer_2": "Morita et al present a manuscript in which they show that bleb-based migration is a common mechanism used by PGCs and cancer cells for extravasation in an in vivo chick embryo system. In short, they show that PGC extravasation is initiated by blebs that are enriched in calcium, measured by the calcium sensor GCaMP6s. Blocking store-operated Ca2+ entry (SOCE) inhibited blebbing and blocked extravasation. In addition, they found that the fibrosarcoma cell line HT-1080 also required SOCE for blebbing and extravasation. Interestingly, two additional epithelial cancer cell lines used calcium increases for blebbing and extravasation, however, they did not utilize SOCE but instead relied on IP₃R-mediated Ca2+ release. The results are interesting, and point toward two related Ca2+ dependent mechanisms for bleb-based TEM, the authors dub them “novel & precise” or “ancient and robust” mechanisms. These findings are important for a better understanding of both developmental processes and cancer metastasis. Strengths of this manuscript include the use of the in vivo chick system for a more complex assessment of TEM than normal in vitro system cannot capitulate, followed by nice mechanistic experiments in an under agarose system. Multiple approaches were taken to disrupt SOCE (pharmacologically and genetically) which adds rigor. This is a solid manuscript and I only have minor points to be addressed-\nMinor points-\n1) It’s unclear how the quantification for the Ca2+ sensor GCaMP6s was done. In the figures, it states “Ratio of GCaMP6s fluorescence”, however, GCaMP6s is not a ratiometric indicator, it is intensity-based. Please clarify how this quantification was done and what the ratio refers to.\n2) Figure 1b the time stamps are incorrect.\n3) Figure 1e and 1h do not specify how many cells were analyzed. There are other instances (such as figure 4c). Please indicate cell numbers in all experiments.\n4) What do the whiskers represent in the box and whisker plots throughout the manuscript? Please add this info to the legends.\n5) The enrichment of ER in the blebs shown in Figure 6 is interesting. How commonly is that seen? The same can be asked about the Orai1 and STIM1 staining (fig 2). Is there any quantification that can be done to indicate how commonly these protein localizations are seen in blebs?", "reviewer_3": "In this study by Morita et al., the authors build on previous work from the lab (Saito et al., 2022) and further characterize the role bleb-based extravasation from the vascular environment of PGCs and cancer tumor cells.\nMore specifically, the authors describe a mechanism promoting extravasation that relies on Ca2+ dependent signaling and favor bleb-based extravasation.\nThe authors combine intravital imaging in chick embryo models for PGC and cancer cell extravasation with state-of-the-art compression tools coupled to live imaging to monitor Ca2+ firing as well as channels dynamics in order to decipher the Ca2+ import and signaling pathways involved in bleb formation and how this promotes extravasation.\nThe role on Ca2+ in extravasation especially in the context of cancer dissemination in not new, the proposed bleb-based extravasation mechanism is quite novel and worthy of interest\nWhile the work contains large amounts of data and combines several state-of-the art approaches, several of the claims are not sufficiently supported by strong quantitative data. Finally, the proposed mechanisms (SOCE versus IP3R) and the associated signaling pathways are not deepened. Besides they are obvious discrepancies between the 2 models and the claims regarding these differences are not supported by data.\nThis prevents the manuscript from being published as it is.\nTo be more specific, the major pitfalls I see in this manuscript are:\n- While the UA compression suggest that confinement is a key mechanical feature driving Ca2+ dependent bleb-like behavior. This also indicates that without compression, there is very little blebbing activity. On the other hand, SCF seems to drive bleb like migration invitro but nothing similar is obviously demonstrated in vivo.\nThe parallel is difficult to draw with in vivo data which show Ca2+ dependent bleb-like protrusion independently of whether cells are constrained or not or independently.\nIt is thus quite difficult to understand what cues are driving such behavior in a relevant vascular context.\n- The characterization of the Ca2+ signaling pathway lacks deepness.\n- Finally, the fact that the mechanism relies on Orai1/STIM1 in certain cell types and IPR3 in others is a bit killing the model.\nThe explanation provided to reconciliate these 2 different mechanisms are thrown a bit abruptly without any experimental evidences.\nBelow are the specific comments on the manuscript:\n1- In figure 1 : the movies aiming at describing the intravascular behavior of PGC are quite are difficult to interpret since :\n• The vascular staining is extremely dotty. A lectin like marker which is widely used for intravital imaging of the vascular environment in many models would have been more suited.\n• The data are supported by still images from LifeAct expressing cells are presented. This probe would have been much more relevant to describe accurately bleb formation. It would have been even more convincing if associated to a membrane probe.\nAlternatively, this could be achieved using chemical probes such as SIRActin or FactAct X and specific membrane/lipidic dyes.\n2- Figure 1C shows obvious alternate actin dependent structures such as filopodia during crawling. Their relevance and role in regard to blebbing are not discussed.\n3- In Figure 2, the authors rly on UA compression to induce\nWhile I do acknowledge and understand that this is an easy way to induce blebbling. It does not recapitulate what is seen in vessels where from the movies and still images shown cells are not compressed.\nIt makes it quite hard to understand what is driving such behavior and is obviously lacking in a manuscript which is centered on extravasation which cannot be uncoupled from the hemodynamic environment.\nA similar comment can be raised from Figure 2f, I – SCF cytokine is used to induce blebbling … but how relevant is it to the in vivo situation?\n4- In figure 2, the authors show that STIM1 is recruited in blebs: however in order to support the model of Ca2+ driven bleb formation supported by Orai1/STIM1, a more dynamic view of the complex is needed. I’m expecting to see STIM1 being recruited before bleb starts. This should be dynamically quantified.\nWithout this, authors cannot exclude that external Ca2+ enters and induces blebs through mechanogated channels like Piezos or TRPs. This should also be test using bread and/or specific drugs such as GMTx4, Dooku … or direct knock down.\n5- Along the same line: is STIM1 recruited with ER ? or ER is already present and populated by STIM1 afterward ?\n6- In Extended data Figure 2e-g, it seems that data are not normalized to a cytosolic FP as it was before. Why is that? This important control should be provided.\n7- SKF96365 is also a TRP inhibitor. This comes back to point to point 4 which should be addressed.\n8- PGCs respond strongly to SCF but not to SDF-1 in the 3D matrigel assay. However, SCF is known to activate several signaling pathways, including PI3K, Src, and PLCγ, and the contribution of these pathways to SOCE activation is not addressed. It is not clear whether SCF activates SOCE directly through STIM/Orai or indirectly via ER depletion through PLCγ.\n9- The authors explore the role of Orai1/STIM1 Ca2+ influx in bleb formation using HT1080, PC3 and MDA-MB-231 which have a rather mesenchymal phenotype. The use of melanoma cell lines which are known to be more ameboid might be relevant and strengthen the data regarding the implication of such mechanism in cancer dissemination.\n10- Orai1 dominant negative is not affecting protrusion formation in HT1080 cells while affecting TEM. This is a bit of a story killer as it is drawing a significant difference with PGC behavior. The authors should provide an explanation as to where is this difference coming from.\n11- Likewise, this pathway has no effect on MDA-MB-231 and PC3. The author show that in this model, Ca2+ relies on IP3R. While the data are convincing, the explanations and claims for the discrepancies are not supported by data.\nMDA-MB-231 an PC3 are mesenchymal like cells that have undergone EMT. They are thus very far from epithelial like cells.\nWhat are the expression levels of ORAI1/STIM1 in the different cells lines?\nHow is this in this different type of cancers? How does expression level relates to cancer aggressiveness. This could be assessed from publicly available transcriptomic data. This would stenghten the claims which are quite weakly supported by data as they stand.\nMinor comments :\n1- L 55 : a bit more context into Ca2+ signaling in bleb initiation might be relevant for a broad audience journal.\n2- In Extended Data Figure 3, the ER-ID signal is highly saturated with dot like structures … this is not very convincing.\n3- The functional suppression with Orai1 E106Q and with IP₃R3 knockdown is interesting, but the study lacks a rescue experiment to fully demonstrate specificity. A re-expression of a siRNA-resistant construct, or at least a quantitative assessment of knockdown efficiency at the protein level, would strengthen the link between the targeted gene and the observed phenotypes.\n4- In several figures in the extended data, it is unclear whether N refers to the number of cells, embryos, or independent biological replicates.\n5- Viability assays are provided for PGCs, but similar controls are not shown for HT-1080, PC-3, or MDA-MB-231 cells. Including viability measurements for these cell lines would help ensure that the observed effects of the inhibitors are not confounded by cytotoxicity."}, "author_response": "Point-by-point responses to the reviewers' comments\nReviewer #1 (Remarks to the Author):\nThis is an interesting paper that uses the avian embryo system as a model to assess the role of blebbing in\nextravasation of PGC cells as well as three cancer cell lines. Using elegant imaging in vivo they\nconvincingly show a role for Ca2+ in bleb formation and extravasation. Surprisingly the source of Ca2+\nchanges based on the cell type, requiring SOCE in PGC and HT-1080 and IP3-dependent Ca2+ release in\nPC1 and MDA231 cells. The functional data is strong and convincing. However, the imaging data for SOCE\neffectors in the bleb is weak and needs to be strengthened. More importantly a more detailed time course\nof the Ca2+ transient, actin polymerization, and bleb formation is critical to better understand the\nmechanisms involved. Currently it is not clear what is the role of the Ca2+ signal, bleb initiation, expansion,\nmaintenance, etc.\n- We sincerely thank Reviewer #1 for the positive and constructive evaluation of our manuscript.\nWe are encouraged that the reviewer found the in vivo imaging and functional perturbation data strong and\nconvincing, and that the study provides an informative comparison of Ca²⁺-dependent bleb-based\nextravasation across PGCs and multiple cancer cell types.\nFirst, to better define the temporal relationship between Ca²⁺ signaling, actin dynamics, and bleb\nmorphogenesis, we added time-resolved kymograph analyses (Extended Data Fig. 2h) and revised the main\ntext accordingly. These analyses show that Ca²⁺ levels remain elevated throughout the bleb expansion–\nretraction cycle, whereas cortical F-actin accumulation temporally correlates with the retraction phase,\nthereby clarifying the likely roles of Ca²⁺ signaling during bleb formation.\nSecond, we carefully reassessed how SOCE-related imaging data are presented and interpreted. We\nadded a split-Venus (STIM1–Orai1) complementation assay demonstrating stimulus-dependent puncta\nformation, including under thapsigargin treatment (Fig. 2e, g), as qualitative but direct evidence for\nSTIM1–Orai1 complex formation in PGCs. The purpose of this experiment was to test whether STIM1–\nOrai1 coupling can be induced under bleb-forming conditions, rather than to provide a quantitative or\nspatially resolved analysis of puncta distribution.\nIn addition, we implemented several clarifications and revisions prompted by the reviewer’s\ncomments, including re-acquisition of ER-ID images under non-saturating conditions (Extended Data Fig.\n3), inclusion of whole-cell Fluo-4 analyses (Extended Data Fig. 9), and refinement of pathway-\ndiscrimination experiments by prioritizing molecule-specific genetic perturbation (IP₃R3 knockdown in\nmain Fig. 6) and using a more selective IP₃R inhibitor (Xestospongin C), with 2-APB data moved to\nExtended Data Fig. 10.\nWe believe that these revisions directly address the reviewer’s concerns and substantially strengthen\nthe manuscript by clarifying the temporal sequence of Ca²⁺ and actin dynamics and by ensuring a\nconservative, evidence-based interpretation of SOCE-related imaging observations.\nSpecific comments:\nIt would be helpful to have timelapse of coincident actin formation and Ca2+ signals within the blebs to\ndetermine their relative time courses.\n- We agree with the reviewer that defining the temporal relationship between Ca²⁺ dynamics and actin\nbehavior within blebs is critical for understanding the mechanism of bleb-based protrusion.\nTo address this point, we performed time-resolved analyses of Ca²⁺ signaling and F-actin dynamics\nduring bleb morphogenesis and added kymograph-based representations for each process (Extended Data\nFig. 2h). These analyses allow direct comparison of the relative timing of Ca²⁺ elevation and actin\nremodeling during the bleb life cycle.\nAccurate quantification of GCaMP6s signals requires normalization to a co-expressed cytosolic\nmCherry control, which precluded simultaneous imaging with Lifeact in the same cell. We therefore\nanalyzed Ca²⁺ dynamics (GCaMP6s) and actin dynamics (Lifeact) in separate but temporally comparable\ntime-lapse datasets acquired under identical experimental conditions.\nThese analyses reveal that Ca²⁺ levels remain elevated throughout the bleb expansion–retraction cycle,\nwhereas cortical F-actin accumulation shows a clear temporal correlation with the retraction phase.\nThis temporal separation supports a model in which Ca²⁺ elevation is associated with bleb initiation and\nmaintenance, while actin reassembly primarily contributes to bleb retraction. These changes are reflected\nin the revised manuscript (Please see Lines 115~).\nExtended figure 3: the STIM1 and Orai1 staining looks normal, however there is an issue with the ER-ID\nstaining not clear what the large blobs in the ER are? Please explain.\n- We thank the reviewer for raising this important point regarding the ER-ID signal in Extended Data Figure\n3.\nER-ID is a qualitative fluorescent marker that reports the overall distribution of the endoplasmic\nreticulum and does not label specific ER proteins or defined molecular or ultrastructural subdomains.\nTherefore, local variations in ER-ID signal intensity cannot be interpreted as evidence for distinct ER\nsubdomains, and we do not infer structural compartmentalization from ER-ID staining.\nIn the original images, STIM1 signals were frequently observed within ER-ID–positive regions that\nappeared to exhibit relatively lower ER-ID intensity. Importantly, this observation does not imply the\npresence of specialized ER subdomains but rather suggests that STIM1 is not uniformly distributed\nthroughout the ER under resting conditions in cultured PGCs, as now explicitly stated in the revised\nmanuscript (Please see Line 129~).\nWe also note that visualization of STIM1 localization initially required relatively high exposure\nsettings for the ER-ID channel, which resulted in partial saturation of strongly labeled ER regions and may\nhave exaggerated the appearance of large ER-ID–positive structures. To address this, we re-acquired ER-\nID images using non-saturating exposure conditions. The revised images confirm that STIM1 localizes to\nER-ID–positive ER compartments independently of ER-ID signal intensity. These updated images have\nreplaced the original ones in the revised Extended Data Figure 3.\nThe functional data supporting a role for SOCE in blebbing in PGCs is strong but the imaging support is\nrather weak. More in depth analysis is required to convincingly show that STIM1-Orai1 junction at formed\nin the blebs. The data in Fig. 2d and 2h are not convincing and actually argue against SOCE activation. The\ndata show ER in the bleb to is close to the plasma membrane consistent with ER-PM contact sites. However,\nSOCE is typically marked by the formation of STIM1-Orai1 puncta. One would need to fix to stabilize the\nblebs and then quantify STIM1-Orai1 puncta indicative of SOCE through z-stacks to be comprehensive\nand compare to the cell body. Also what happens to these puncta under thapsigargin treatment.\n- We thank the reviewer for this important and constructive comment regarding the imaging-based evidence\nfor SOCE activation in PGCs. We agree that the ER–plasma membrane proximity shown in Fig. 2d and 2h\nalone is not sufficient to demonstrate SOCE activation, as SOCE is classically defined by stimulus-\ndependent STIM1–Orai1 coupling and puncta formation rather than ER–PM apposition per se.\nTo directly test whether STIM1–Orai1 interactions can be induced under bleb-forming conditions, we\nemployed a split-Venus complementation approach using STIM1–VC and Orai1–VN constructs. This\nsystem reports physical proximity between STIM1 and Orai1 through irreversible Venus fluorescence\nreconstitution and has been previously validated as a qualitative readout of SOCE complex assembly in\nliving cells (https://link.springer.com/protocol/10.1007/978-1-4939-7759-8_16).\nUsing this approach, we observed stimulus-dependent formation of discrete Venus puncta following\nbleb-inducing conditions, including agarose compression, as well as after thapsigargin treatment to forcibly\nactivate SOCE. Such puncta were absent under non-stimulated conditions. These observations provide\ndirect evidence that STIM1–Orai1 coupling can be induced in PGCs under conditions that promote bleb\nformation. Please see Lines 138~, Line 152~ and Line 615~ for the corresponding revisions in the Results\nand Materials and Methods.\nWe acknowledge that this split-Venus approach does not allow rigorous quantitative or spatial\nanalysis of puncta distribution within blebs. Because Venus reconstitution is irreversible, the signal cannot\nreport dynamic puncta turnover, and variable transfection efficiency in PGCs precludes reliable comparison\nof puncta number or density between blebs and the cell body. While fixation can stabilize bleb morphology\nin some systems, in our experimental context the primary limitations arise from the irreversible nature of\nthe reporter and expression variability rather than from fixation itself.\nAccordingly, we interpret the split-Venus data as qualitative but direct evidence for stimulus-\ndependent STIM1–Orai1 complex formation, rather than as a comprehensive spatial or quantitative\ncharacterization of SOCE junctions. Importantly, our conclusions regarding SOCE-dependent bleb\nformation and extravasation are based primarily on convergent functional perturbation experiments\n(genetic and pharmacological), with the imaging data serving as supportive evidence.\nAre GCAMP measurements in the blebs rapid enough to detect Ca2+ entry through SOCE?\n- We believe that the temporal resolution of GCaMP6s is sufficient to detect Ca²⁺ entry associated with\nSOCE in our experimental context. SOCE-mediated Ca²⁺ influx occurs on the timescale of seconds to tens\nof seconds, which is well within the response kinetics of GCaMP6s.\nIn our live-imaging analyses, we consistently observed clear and reproducible enrichment of\nGCaMP6s fluorescence within blebs compared to the adjacent cell body during bleb formation. These Ca²⁺\nsignals were spatially restricted to the bleb region and persisted throughout the bleb expansion–retraction\ncycle, indicating sustained Ca²⁺ elevation rather than transient noise. Importantly, we confirmed that these\nbleb-associated Ca²⁺ signals were abolished by extracellular Ca²⁺ chelation and by pharmacological\ninhibition of SOCE, supporting the conclusion that GCaMP6s reliably reports Ca²⁺ entry events relevant to\nSOCE under our imaging conditions.\nConsistent with our approach, GCaMP6s has been successfully used in previous studies to visualize\nCa²⁺ dynamics associated with bleb formation and membrane–cortex detachment, including in systems\nwhere Ca²⁺ influx occurs over comparable temporal scales (e.g., Nature Communications, 2021:\nhttps://www.nature.com/articles/s41467-020-20826-5).\nThus, while GCaMP6s may not resolve single-channel opening events, its sensitivity and temporal\nresolution are sufficient to capture the functionally relevant Ca²⁺ dynamics associated with SOCE-\ndependent bleb formation in this study.\nFor the fluo4 measurements in Fig. 5 show the entire cell as it is not clear what the levels of Ca2+ are across\nthe entire cell compared to the bleb.\n- In response to this comment, we added whole-cell Fluo-4 imaging in Extended Data Figure 9, enabling\ndirect comparison of Ca²⁺ levels between blebs and the cell body. These data confirm that Ca²⁺ signals are\nspatially enriched in blebs during bleb formation.\n2-APB blocks both SOCE and the IP3R so it is not a good drug to separate the two pathways. Better use a\nmore specific IP3R blocker to support the knockdown experiments. The knockdown data are much stronger\nand more convincing so consider moving them into the main figures while removing the pharmacology\ndata.\n- We replaced 2-APB with the more selective IP₃R inhibitor Xestospongin C and moved the 2-APB data to\nExtended Data Figure 10. In addition, we prioritized molecular specificity by moving IP₃R3 knockdown\nexperiments to the main Figure 6, where they now provide the primary evidence for the role of IP₃R-\ndependent Ca²⁺ release. Please see Lines 331~ and 338~ for the corresponding revisions in the Results.\nReviewer #2 (Remarks to the Author):\nMorita et al present a manuscript in which they show that bleb-based migration is a common mechanism\nused by PGCs and cancer cells for extravasation in an in vivo chick embryo system. In short, they show\nthat PGC extravasation is initiated by blebs that are enriched in calcium, measured by the calcium sensor\nGCaMP6s. Blocking store-operated Ca2+ entry (SOCE) inhibited blebbing and blocked extravasation.\nIn addition, they found that the fibrosarcoma cell line HT-1080 also required SOCE for blebbing and\nextravasation. Interestingly, two additional epithelial cancer cell lines used calcium increases for\nblebbing and extravasation, however, they did not utilize SOCE but instead relied on IP₃R-mediated Ca2+\nrelease. The results are interesting, and point toward two related Ca2+ dependent mechanisms for bleb-based TEM,\nthe authors dub them “novel & precise” or “ancient and robust” mechanisms. These findings are important\nfor a better understanding of both developmental processes and cancer metastasis. Strengths of this\nmanuscript include the use of the in vivo chick system for a more complex assessment of TEM than normal\nin vitro system cannot capitulate, followed by nice mechanistic experiments in an under agarose system.\nMultiple approaches were taken to disrupt SOCE (pharmacologically and genetically) which adds rigor.\nThis is a solid manuscript and I only have minor points to be addressed-\n- We sincerely thank the reviewer for the positive and thoughtful assessment of our manuscript. We are\nencouraged that the reviewer finds the study solid and that our findings provide meaningful insight into\nCa²⁺-dependent mechanisms of bleb-based transendothelial migration in development and cancer.\nWe have addressed all minor points raised, as detailed below, and believe these revisions further\nstrengthen the manuscript.\nMinor points-\n1) It’s unclear how the quantification for the Ca2+ sensor GCaMP6s was done. In the figures, it states “Ratio\nof GCaMP6s fluorescence”, however, GCaMP6s is not a ratiometric indicator, it is intensity-based. Please\nclarify how this quantification was done and what the ratio refers to.\n- We thank the reviewer for pointing out this ambiguity and apologize for the lack of clarity. GCaMP6s is\nindeed an intensity-based Ca²⁺ indicator and not a ratiometric probe in the strict sense.\nIn all quantifications, GCaMP6s fluorescence intensity was normalized to the fluorescence intensity\nof co-expressed cytoplasmic mCherry within the same cell. Thus, the values represent relative GCaMP6s\nfluorescence (GCaMP6s/mCherry), rather than a true ratiometric Ca²⁺ measurement.\nTo avoid confusion, we have revised the y-axis labeling in the relevant figures from “Ratio of\nGCaMP6s fluorescence” to “Relative fluorescence of GCaMP6s (normalized to mCherry)” and have\nexplicitly clarified this normalization procedure in the corresponding figure legends (Fig. 1e,h) and\nMaterials and Methods (Please see Lines 865~).\n2) Figure 1b the time stamps are incorrect.\n- We thank the reviewer for noticing this error. The time stamps in Figure 1b have been corrected in the\nrevised manuscript.\n3) Figure 1e and 1h do not specify how many cells were analyzed. There are other instances (such as figure\n4c). Please indicate cell numbers in all experiments.\n- We thank the reviewer fir this helpful comment. We have now indicated the number of cells analyzed in\nFigure 1e, Figure 1h, Figure 4c, and all other relevant figure legends throughout the manuscript.\n4) What do the whiskers represent in the box and whisker plots throughout the manuscript? Please add this\ninfo to the legends.\n- We thank the reviewer for this clarification request. In all box-and-whisker plots, the center line represents\nthe median, the box indicates the interquartile range (25th–75th percentiles), and the whiskers extend to the\nminimum and maximum values. We have now explicitly added this information to the figure legends and\nthe Methods section for clarity (Please see Line 920~).\n5) The enrichment of ER in the blebs shown in Figure 6 is interesting. How commonly is that seen? The\nsame can be asked about the Orai1 and STIM1 staining (fig 2). Is there any quantification that can be done\nto indicate how commonly these protein localizations are seen in blebs?\n- We thank the reviewer for this insightful comment. Based on our observations, ER entry into blebs is a\nhighly reproducible feature across blebbing events. In the vast majority of bleb-forming cells analyzed, ER\nstructures were consistently detected within blebs.\nWe further noted clear cell-type–specific differences in the morphology of ER extensions within blebs,\nwhich are now described in the Results. In PC-3 cells, ER invasion was highly stereotyped: in all blebs\nanalyzed, two or more ER tubules entered individual blebs. In contrast, MDA-MB-231 cells showed more\nheterogeneous ER morphologies, with a single ER filament observed in 69.2% of blebs and two or more\nER filaments in 30.8% of blebs. In SOCE-dependent cells, including chick PGCs and HT-1080 cells, ER\ninvasion patterns more closely resembled those observed in PC-3 cells, with multiple thin ER extensions\nfrequently entering blebs.\nA similar consideration applies to the localization of STIM1 and Orai1. Enrichment of these proteins\nin bleb-associated regions was reproducibly observed under bleb-inducing conditions. However, rigorous\nquantitative assessment of their spatial distribution within blebs remains technically challenging. Bleb\nformation is a rapid, highly dynamic, and three-dimensional process, and accurate quantification would\nrequire high-speed volumetric imaging with sufficient axial resolution, which is beyond the scope of the\npresent study. For these reasons, we present ER, STIM1, and Orai1 localization within blebs primarily in a\nqualitative manner and avoid overinterpretation. Please see Line 320~ for the corresponding revisions in\nthe Results.\nReviewer #3 (Remarks to the Author):\nIn this study by Morita et al., the authors build on previous work from the lab (Saito et al., 2022) and further\ncharacterize the role bleb-based extravasation from the vascular environment of PGCs and cancer tumor\ncells. More specifically, the authors describe a mechanism promoting extravasation that relies on Ca2+\ndependent signaling and favor bleb-based extravasation.\nThe authors combine intravital imaging in chick embryo models for PGC and cancer cell extravasation with\nstate-of-the-art compression tools coupled to live imaging to monitor Ca2+ firing as well as channels\ndynamics in order to decipher the Ca2+ import and signaling pathways involved in bleb formation and how\nthis promotes extravasation. The role on Ca2+ in extravasation especially in the context of cancer\ndissemination in not new, the proposed bleb-based extravasation mechanism is quite novel and worthy of\ninterest. While the work contains large amounts of data and combines several state-of-the art approaches,\nseveral of the claims are not sufficiently supported by strong quantitative data. Finally, the proposed\nmechanisms (SOCE versus IP3R) and the associated signaling pathways are not deepened. Besides they\nare obvious discrepancies between the 2 models and the claims regarding these differences are not\nsupported by data. This prevents the manuscript from being published as it is.\nTo be more specific, the major pitfalls I see in this manuscript are:\n- While the UA compression suggest that confinement is a key mechanical feature driving Ca2+ dependent\nbleb-like behavior. This also indicates that without compression, there is very little blebbing activity. On\nthe other hand, SCF seems to drive bleb like migration invitro but nothing similar is obviously demonstrated\nin vivo.\nThe parallel is difficult to draw with in vivo data which show Ca2+ dependent bleb-like protrusion\nindependently of whether cells are constrained or not or independently.\nIt is thus quite difficult to understand what cues are driving such behavior in a relevant vascular context.\n- The characterization of the Ca2+ signaling pathway lacks deepness.\n- Finally, the fact that the mechanism relies on Orai1/STIM1 in certain cell types and IPR3 in others is a\nbit killing the model. The explanation provided to reconciliate these 2 different mechanisms are thrown a\nbit abruptly without any experimental evidences.\n- We thank Reviewer #3 for the careful, detailed, and constructive evaluation of our manuscript, and for\nappreciating the conceptual significance of bleb-based extravasation as a Ca²⁺-dependent mechanism\noperating in both developmental and cancer contexts.\nWe agree that the involvement of Ca²⁺ signaling in cancer dissemination has been reported previously, and\nwe also acknowledge the importance of comprehensively reconstructing upstream physiological cues and\nthe entire Ca²⁺ signaling network acting in vivo. However, the primary objective of the present study was\nnot to exhaustively dissect all upstream inputs. Rather, our aim was to identify and experimentally validate\na Ca²⁺-dependent bleb-forming mechanism that functions during extravasation in a physiologically relevant\nin vivo environment.\nUsing intravital imaging in avian embryos, we demonstrate that localized Ca²⁺ elevation within bleb-like\nprotrusions occurs in parallel with extravasation, and that perturbation of Ca²⁺ supply robustly suppresses\nboth bleb formation and transendothelial migration. Importantly, we interpret SOCE-dependent Ca²⁺ entry\nand IP₃R-dependent Ca²⁺ release as distinct Ca²⁺ mobilization routes that, despite their differences, operate\nat the same functional level and ultimately converge on a shared downstream process—bleb formation\ndriven by local Ca²⁺ elevation. Providing this conceptual framework was a central objective of the study.\nWe apologize that this intention was not communicated clearly\nenough in the previous version of the manuscript. To avoid any\nmisunderstanding that these distinct Ca²⁺ pathways might be\ninterpreted as “killing the model,” we now share a conceptual\nframework illustrating this hierarchical relationship, in which\ndifferent Ca²⁺ recruitment mechanisms are engaged depending on\ncell type and context, yet converge on a common Ca²⁺-dependent\nbleb-forming program (see schematic model). We further envisage\nthat upstream of these Ca²⁺ mobilization routes, multiple and\npotentially divergent stimuli—such as mechanical cues and growth factor signaling—may contribute. We\nregret that the earlier presentation may have given the impression that the coexistence of these Ca²⁺\npathways undermines the coherence of the model; our intention is instead to emphasize their functional\nconvergence.\nWe fully recognize that detailed identification of upstream physiological triggers and comprehensive\nreconstruction of the Ca²⁺ signaling network are highly important and meaningful goals. However, we\nconsider these analyses to be beyond the scope of the present study. These issues are addressed in more\ndetail in our responses to the specific comments below, where we provide additional analyses and\nclarifications. We believe that the revisions made have placed our conclusions within a clearer and\nexperimentally supported framework, thereby strengthening the manuscript.\nFinally, we are grateful for these comments, as they prompted us to re-examine and more clearly articulate\nour central claims, and also led to the development of a more meaningful and focused discussion.\nBelow are the specific comments on the manuscript:\n1- In figure 1 : the movies aiming at describing the intravascular behavior of PGC are quite are difficult to\ninterpret since :\n• The vascular staining is extremely dotty. A lectin like marker which is widely used for intravital imaging\nof the vascular environment in many models would have been more suited.\n• The data are supported by still images from LifeAct expressing cells are presented. This probe would have\nbeen much more relevant to describe accurately bleb formation. It would have been even more convincing\nif associated to a membrane probe.\nAlternatively, this could be achieved using chemical probes such as SIRActin or FactAct X and specific\nmembrane/lipidic dyes.\n- We thank the reviewer for the constructive comments regarding visualization of intravascular PGC\nbehavior. Regarding vascular labeling, we would like to clarify that the choice of marker was based on\nextensive prior testing. In our previous study (Developmental Biology, 2025;\nhttps://www.sciencedirect.com/science/article/pii/S0012160625001332), we systematically evaluated\nmultiple vascular labeling strategies in both live and fixed avian embryos. Based on these analyses, we\nconcluded that acetylated low-density lipoprotein (AcLDL) represents the most suitable option for live\nimaging, as it does not induce blood flow stagnation or cellular aggregation.\nWe also tested lectin-based labeling using Lens culinaris lectin, which indeed provides highly detailed\nendothelial staining. However, in live embryos this lectin consistently caused severe cell aggregation and\nimpaired circulation, rendering it unsuitable for intravital imaging (while remaining applicable to fixed\nsamples). For this reason, AcLDL was selected for vascular labeling in live imaging experiments in the\npresent study.\nWith respect to bleb visualization, the primary aim of Figure 1 was to document the in vivo intravascular\ndynamics and spatial positioning of PGCs. To minimize phototoxicity and perturbation of fragile cellular\nbehaviors during long-term live imaging, we therefore used representative still images from LifeAct-\nexpressing cells to illustrate actin cortex remodeling associated with bleb formation.\nWe agree that combining membrane probes or chemical dyes (such as SiR-Actin, F-tractin variants, or lipid\ndyes) with actin reporters would be informative. However, in early avian embryos, long-term intravital\nimaging using such probes is technically challenging due to limitations in probe delivery, signal stability,\nand phototoxicity. Consequently, we adopted a strategy combining genetically encoded reporters with\ncomplementary still images.\nImportantly, bleb formation itself was independently validated throughout the study using a series of in\nvitro assays, in which membrane deformation and actin dynamics can be analyzed at higher spatial\nresolution. These points are now explicitly clarified in the revised Results section.\n2- Figure 1C shows obvious alternate actin dependent structures such as filopodia during crawling. Their\nrelevance and role in regard to blebbing are not discussed.\n- We thank the reviewer for this insightful observation. We agree that actin-dependent protrusive structures\nsuch as filopodia are visible in some crawling PGCs and may play important roles in cell migration under\ncertain contexts.\nHowever, the primary focus of the present study is to characterize bleb-based protrusions during\nintravascular arrest and transendothelial migration, rather than to comprehensively analyze all modes of\nactin-driven motility. For this reason, we deliberately limit our mechanistic discussion to bleb-associated\nactin remodeling and avoid extending interpretation to other protrusive structures that were not\nsystematically analyzed.\nImportantly, the presence of filopodia-like structures does not contradict our conclusions. We have now\nadded a brief note in the manuscript indicating that actin-rich filopodia can also be observed during crawling\n(Please see Line 96).\n3- In Figure 2, the authors rely on UA compression to induce\nWhile I do acknowledge and understand that this is an easy way to induce blebbing. It does not recapitulate\nwhat is seen in vessels where from the movies and still images shown cells are not compressed.\nIt makes it quite hard to understand what is driving such behavior and is obviously lacking in a manuscript\nwhich is centered on extravasation which cannot be uncoupled from the hemodynamic environment.\nA similar comment can be raised from Figure 2f, I – SCF cytokine is used to induce blebbing … but how\nrelevant is it to the in vivo situation?\n- We thank the reviewer for raising this fundamental point.\nComments 3 and 8 relate to a shared and fundamental question regarding the upstream mechanisms that\ntrigger bleb formation under physiological conditions.\nWe fully agree that under-agarose compression and SCF stimulation do not directly recapitulate the\nmechanical or hemodynamic environment experienced by cells within blood vessels in vivo.\nIn this study, these paradigms were not intended to model the vascular environment per se, but rather to\nserve as operational assays that reproducibly induce bleb formation observed during in vivo extravasation.\nThis allowed us to functionally dissect the Ca²⁺ dependence and molecular requirements of bleb-based\nprotrusions, independent of the precise upstream trigger.\nImportantly, we directly demonstrate that both PGCs and cancer cells form Ca²⁺-enriched blebs and\nundergo transendothelial migration in vivo, even in the absence of obvious external compression. Thus, the\ncentral question addressed here is whether Ca²⁺ elevation is sufficient to drive bleb formation and\nextravasation once initiated, rather than identifying the ultimate physiological trigger.\nWe now explicitly clarify this distinction in the Discussion and highlight the identification of in vivo\nupstream cues—such as shear stress, endothelial interactions, and cytokine gradients—as an important\ntopic for future investigation.\n4- In figure 2, the authors show that STIM1 is recruited in blebs: however in order to support the model of\nCa2+ driven bleb formation supported by Orai1/STIM1, a more dynamic view of the complex is needed.\nI’m expecting to see STIM1 being recruited before bleb starts. This should be dynamically quantified.\nWithout this, authors cannot exclude that external Ca2+ enters and induces blebs through mechanogated\nchannels like Piezos or TRPs. This should also be test using bread and/or specific drugs such as GMTx4,\nDooku … or direct knock down.\n- We thank the reviewer for raising this important mechanistic question regarding the temporal role of\nSOCE in bleb formation and the potential contribution of mechanosensitive Ca²⁺ channels.\nA key issue raised is whether SOCE activity contributes causally to the initiation of bleb formation.\nWe agree that a fully quantitative demonstration of STIM1 recruitment strictly preceding bleb onset would\nbe highly informative. However, such an analysis is technically challenging due to the rapid, three-\ndimensional nature of bleb formation and the difficulty of simultaneously resolving STIM1 dynamics,\nCa²⁺ transients, and membrane deformation at sufficiently high temporal resolution in vivo.\nRather than relying solely on temporal correlation, we therefore addressed causality using a\nconvergent functional framework. First, in the revised analyses, pharmacological blockade of SOCE using\nBTP2, a selective inhibitor of STIM-dependent SOCE, completely abolished detectable bleb initiation in\nPGCs, indicating that SOCE activity is required for bleb formation under these conditions (Please see Line\n147). Second, we further tested whether SOCE activation is sufficient to induce blebbing by depleting ER\nCa²⁺ stores with thapsigargin—an established activator of SOCE—which robustly induced bleb formation\neven in the absence of mechanical confinement (Please see Line 150). Under these thapsigargin-induced\nconditions, stimulus-dependent STIM1–Orai1 association was directly detected using a split-Venus\ncomplementation assay, providing molecular evidence for SOCE complex formation during bleb induction\n(Please see Line 152). Third, consistent with our original observations, direct elevation of intracellular Ca²⁺\nusing the Ca²⁺ ionophore A23187 was likewise sufficient to trigger blebbing (Please see Line 153).\nTogether, these loss-of-function and gain-of-function experiments support a model in which SOCE-\nmediated Ca²⁺ entry plays a causal role in the initiation of bleb formation in this experimental context,\neven if precise temporal ordering cannot be resolved at the level of individual molecular events.\nThe reviewer also raises the possibility that mechanosensitive Ca²⁺ channels such as TRP or Piezo\nchannels may contribute to bleb initiation. While we do not formally exclude a contributory role for such\nchannels, the complete suppression of bleb initiation by low-dose BTP2 argues against a dominant\ncontribution of mechanosensitive Ca²⁺ entry under our experimental conditions.\nWe have revised the manuscript accordingly to clarify this functional framework and to incorporate\nthe additional molecular and functional evidence supporting a requirement for SOCE in bleb initiation\n(Please see Line 147 and 152).\n5- Along the same line: is STIM1 recruited with ER? or ER is already present and populated by STIM1\nafterward?\n- We thank the reviewer for this thoughtful question regarding the relationship between ER dynamics and\nSTIM1 recruitment during bleb formation.\nIn the canonical model of store-operated Ca²⁺ entry, STIM1 is an ER-resident protein that redistributes\nwithin pre-existing ER structures in response to Ca²⁺ store depletion, becoming enriched at ER regions\nclosely apposed to the plasma membrane to form ER–PM contact sites. Within this framework, the key\nevent is not the de novo recruitment of ER, but rather the local accumulation of STIM1 within specific ER\nsubdomains adjacent to the plasma membrane (https://rupress.org/jcb/article/174/6/803/44527/Ca2-store-\ndepletion-causes-STIM1-to-accumulate-in).\nAccordingly, in the context of our study, the critical issue is not the strict temporal ordering of ER entry\nversus STIM1 recruitment, but whether STIM1-dependent SOCE activity is functionally coupled to bleb\nformation. Resolving the precise spatiotemporal sequence of ER morphology, STIM1 redistribution, Ca²⁺\nsignaling, and membrane deformation during the very early phase of bleb initiation would require\nsimultaneous high-speed, three-dimensional imaging of all these parameters. At present, such\nmeasurements exceed the capabilities of our experimental setup.\nImportantly, our central conclusions do not depend on the exact temporal ordering of ER presence and\nSTIM1 enrichment. In the revised manuscript, we clarify this conceptual framework and restrict discussion\nof ER–STIM1 dynamics to observations that can be directly supported by the present data.\n6- In Extended data Figure 2e-g, it seems that data are not normalized to a cytosolic FP as it was before.\nWhy is that? This important control should be provided.\n- We thank the reviewer for pointing this out and apologize for the lack of clarity.\nAs in other Ca²⁺ imaging experiments throughout the manuscript, the data shown in Extended Data Figure\n2e–g were normalized to a co-expressed cytosolic fluorescent protein (mCherry) to account for differences\nin expression level, cell thickness, and imaging conditions.\nThis normalization was inadvertently not stated explicitly in the figure legend. We have now corrected this\nby clearly indicating the GCaMP6s/mCherry normalization in the revised figure legend (Fig. 1e,h) and\nMethods section, consistent with the quantification described elsewhere in the manuscript (Please see Lines\n865~).\n7- SKF96365 is also a TRP inhibitor. This comes back to point to point 4 which should be addressed.\n- We agree with the reviewer that SKF96365 is not specific to SOCE and can also inhibit TRP channels,\nand therefore this concern is closely related to point 4.\nTo address this issue, we performed additional experiments using BTP2, which is widely used as a more\nselective inhibitor of STIM1-dependent store-operated Ca²⁺ entry. As shown in the newly added Figure\n2a,b, BTP2 robustly suppressed bleb initiation in PGCs at concentrations lower than those required for\nSKF96365.\nImportantly, at these low concentrations BTP2 is reported to have minimal effects on TRP channels,\narguing against a dominant contribution of TRP-mediated Ca²⁺ entry. These additional data therefore\nstrengthen our conclusion that SOCE activity is required for bleb initiation and directly address the concern\nraised here and in point 4.\n8- PGCs respond strongly to SCF but not to SDF-1 in the 3D matrigel assay. However, SCF is known to\nactivate several signaling pathways, including PI3K, Src, and PLCγ, and the contribution of these pathways\nto SOCE activation is not addressed. It is not clear whether SCF activates SOCE directly through\nSTIM/Orai or indirectly via ER depletion through PLCγ.\n- We thank the reviewer for this insightful and mechanistically important question.\nWe agree that SCF activates multiple downstream signaling pathways, including PI3K, Src, and PLCγ, and\nthat PLCγ-mediated ER Ca²⁺ depletion could in principle lead to indirect activation of SOCE via STIM/Orai\ncoupling. Dissecting whether SCF engages SOCE through direct signaling to STIM/Orai complexes or\nindirectly through ER Ca²⁺ store depletion represents an important mechanistic distinction.\nHowever, as clarified in response to Comments 3, the aim of the present study was not to resolve the precise\nupstream signaling cascade that initiates bleb formation, but rather to functionally test whether Ca²⁺-\ndependent blebbing and migration require SOCE activity once triggered. In this context, we show that\nperturbation of SOCE components robustly suppresses SCF-induced blebbing and migration,\ndemonstrating that SOCE is functionally required downstream of SCF stimulation, irrespective of the exact\nmode of activation.\nWe now explicitly state in the Discussion that the upstream mechanisms by which SCF engages Ca²⁺\nsignaling, including the potential involvement of PLCγ-mediated ER Ca²⁺ depletion, remain unresolved\nand represent an important direction for future investigation.\n9- The authors explore the role of Orai1/STIM1 Ca2+ influx in bleb formation using HT1080, PC3 and\nMDA-MB-231 which have a rather mesenchymal phenotype. The use of melanoma cell lines which are\nknown to be more ameboid might be relevant and strengthen the data regarding the implication of such\nmechanism in cancer dissemination.\n- We thank the reviewer for this important suggestion.\nUpon reconsideration, we agree that our previous discussion may have overextended the interpretation by\nimplying a direct link between cancer cell origin (epithelial versus mesenchymal) and Ca²⁺ mobilization\nmechanisms, which goes beyond the experimental scope of the present study.\nAccordingly, we have revised the final part of the Discussion to remove speculative statements linking cell\nlineage or EMT status to specific Ca²⁺ recruitment pathways. Instead, we now emphasize as a future\ndirection the need for systematic analyses across a broader range of cancer types with differing origins and\ndegrees of malignancy to determine how generalizable these Ca²⁺ mobilization strategies are.\n10- Orai1 dominant negative is not affecting protrusion formation in HT1080 cells while affecting TEM.\nThis is a bit of a story killer as it is drawing a significant difference with PGC behavior. The authors should\nprovide an explanation as to where is this difference coming from.\n- We thank the reviewer for raising this important point. Extended Data Fig. 8g,h was designed to evaluate\nthe impact of dominant-negative Orai1 on actin-rich protrusions (filopodia-like structures), and indeed\nthese protrusions are largely preserved, indicating that Orai1-dependent Ca²⁺ entry is dispensable for\nfilopodial protrusion formation in HT-1080 cells.\nImportantly, this does not contradict the robust reduction in blebbing and TEM/extravasation upon\nSOCE inhibition shown in the main figures. Rather, we interpret the data as evidence for protrusive\nredundancy in cancer cells: HT-1080 cells can utilize both bleb-based and actin-rich (filopodial/hybrid)\nprotrusions during extravasation. Under Orai1 inhibition, the bleb-based route is strongly impaired, while\na filopodia-enabled route may remain available, providing a straightforward explanation for why\nextravasation is reduced but not completely abolished.\nIn contrast, PGCs predominantly rely on bleb-based protrusive activity for transendothelial passage,\nmaking their extravasation more uniformly sensitive to SOCE perturbation. We have revised the main text\nto explicitly distinguish actin-rich protrusions from bleb-based protrusions and to incorporate the\ninterpretation that, under Orai1 inhibition, a filopodia-enabled route may remain available in cancer cells,\nproviding a straightforward explanation for why extravasation is reduced but not completely abolished.\nThese revisions clarify the cell-type-specific protrusive strategies underlying TEM (Please see Line 266).\n11- Likewise, this pathway has no effect on MDA-MB-231 and PC3. The author show that in this model,\nCa2+ relies on IP3R. While the data are convincing, the explanations and claims for the discrepancies are\nnot supported by data.\nMDA-MB-231 and PC3 are mesenchymal like cells that have undergone EMT. They are thus very far from\nepithelial like cells.\n- We appreciate the reviewer’s comment and agree that our original Discussion may have overextended the\ninterpretation of the observed differences in Ca²⁺ mobilization mechanisms. In particular, we acknowledge\nthat linking SOCE- or IP₃R-dependence to cellular origin or EMT status is not directly supported by the\ndata presented in this study. We have therefore revised the Discussion following the same rationale\ndescribed in our response to Comment 9.\nWhat are the expression levels of ORAI1/STIM1 in the different cell lines?\nHow is this in this different type of cancers? How does expression level relates to cancer aggressiveness.\nThis could be assessed from publicly available transcriptomic data. This would strengthen the claims which\nare quite weakly supported by data as they stand.\n- We thank the reviewer for this helpful and constructive suggestion.\nTo assess whether differential usage of SOCE- versus IP₃R-dependent Ca²⁺ mobilization could be explained\nby expression levels, we analyzed publicly available RNA-seq data from the Cancer Cell Line Encyclopedia\n(CCLE). Specifically, we compared transcript levels of key SOCE components (STIM1, STIM2, ORAI1,\nORAI2, ORAI3) and IP₃ receptor genes (including ITPR3) across the cancer cell lines used in this study\n(Please see Table 2).\nImportantly, these analyses did not reveal clear expression patterns that segregate SOCE-dependent cells\n(HT-1080) from IP₃R-dependent cells (PC-3 and MDA-MB-231). Thus, the observed divergence in Ca²⁺\nrecruitment mechanisms cannot be explained simply by basal transcript abundance of these channels or\nreceptors.\nThis result supports the conclusion that Ca²⁺ supply strategy is determined not by channel expression levels\nper se, but by functional allocation of Ca²⁺ signaling pathways—potentially involving differences in\nchannel coupling, subcellular organization, or pathway usage—rather than quantitative expression.\nWhile the relationship between SOCE/IP₃R expression and cancer aggressiveness is an important and\ninteresting question, a systematic analysis across tumor types and clinical contexts is beyond the scope of\nthe present study. We now explicitly frame this point as a future research direction in the revised Discussion,\nstating that broader comparative analyses across tumor types, cellular contexts, and degrees of malignancy\nwill be required to determine how Ca²⁺ mobilization strategies are selected and diversified.\nMinor comments :\n1- L 55 : a bit more context into Ca2+ signaling in bleb initiation might be relevant for a broad audience\njournal.\n- We thank the reviewer for this helpful suggestion. We agree that providing additional context on Ca²⁺\nsignaling in bleb formation would improve accessibility for a broad readership.\nIn the revised manuscript, we have expanded the Introduction (around line 55) to briefly outline how Ca²⁺\nsignaling has been implicated in the early phase of bleb formation across multiple cellular systems. This\nadded context positions bleb-based protrusions within the broader framework of Ca²⁺-regulated cell\nmechanics, while maintaining the focus of the study on in vivo extravasation.\n2- In Extended Data Figure 3, the ER-ID signal is highly saturated with dot like structures … this is not\nvery convincing.\n- We thank the reviewer for this comment. This issue was also raised by Reviewer #1.\nIn the revised manuscript, we have adjusted the imaging settings and presentation of the ER-ID signal to\navoid saturation and excessive punctate appearance. The corresponding panels in Extended Data Figure 3\nhave been replaced with optimized images that more accurately reflect ER morphology. These changes\nimprove clarity and address the reviewer’s concern.\n3- The functional suppression with Orai1 E106Q and with IP₃R3 knockdown is interesting, but the study\nlacks a rescue experiment to fully demonstrate specificity. A re-expression of a siRNA-resistant construct,\nor at least a quantitative assessment of knockdown efficiency at the protein level, would strengthen the link\nbetween the targeted gene and the observed phenotypes.\n- We thank the reviewer for this constructive suggestion. To address specificity, we focused on minimizing\npotential off-target effects and employing convergent perturbation strategies rather than introducing\nadditional genetic manipulations.\nFor IP₃R3, we used two independent siRNAs targeting distinct regions of the ITPR3 transcript, both\nof which yielded consistent phenotypic outcomes. Efficient suppression of ITPR3 expression was\nquantitatively confirmed at the mRNA level by qPCR, as now explicitly stated in the revised manuscript\n(please see Line 335). We note that, given the concordance between independent siRNAs and the robustness\nof the resulting phenotypes, additional rescue experiments were not pursued.\nFor Orai1, we employed a dominant-negative pore mutant (Orai1 E106Q), which directly disrupts\nchannel function and has been widely used to specifically inhibit SOCE activity. Because this strategy\ninterferes with channel function at the pore level, it does not readily lend itself to a conventional rescue\ndesign. Instead, specificity is supported by the strong concordance between dominant-negative inhibition,\npharmacological SOCE blockade, and the resulting functional phenotypes.\nTogether, these complementary genetic and pharmacological perturbations provide convergent\nevidence that the observed effects are specifically attributable to disruption of IP₃R3- or SOCE-dependent\nCa²⁺ signaling, even in the absence of additional rescue experiments.\n4- In several figures in the extended data, it is unclear whether N refers to the number of cells, embryos, or\nindependent biological replicates.\n- We thank the reviewer for pointing this out. We have now clarified in all figure legends whether N refers\nto the number of cells, embryos, or independent biological replicates.\n5- Viability assays are provided for PGCs, but similar controls are not shown for HT-1080, PC-3, or MDA-\nMB-231 cells. Including viability measurements for these cell lines would help ensure that the observed\neffects of the inhibitors are not confounded by cytotoxicity.\n- We thank the reviewer for raising this important point.\nTo address the possibility that the observed effects on bleb formation and extravasation could be\nconfounded by cytotoxicity, we have now included viability analyses for additional cancer cell lines in the\nrevised manuscript.\nSpecifically, we assessed cell viability following expression of dominant-negative Orai1 (Orai1 E106Q) in\nPC-3 and MDA-MB-231 cells. Notably, both cell lines exhibited a marked reduction in viability within 24\nhours of dominant-negative Orai1 expression. In contrast, the same manipulation did not compromise\nviability in PGCs or HT-1080 cells.\nThese results indicate that, unlike PGCs and HT-1080 cells, PC-3 and MDA-MB-231 cells critically depend\non SOCE for cell survival. This does not indicate that bleb formation is Ca²⁺-independent in these cells.\nRather, it suggests that SOCE-mediated Ca²⁺ influx is functionally allocated to maintaining cell viability,\nwhereas bleb formation relies on alternative Ca²⁺ sources.\nConsistent with this interpretation, bleb formation and extravasation in PC-3 and MDA-MB-231 cells rely\npredominantly on IP₃R-dependent Ca²⁺ release, as demonstrated by our pharmacological and genetic\nperturbation experiments. Thus, the absence of SOCE-dependent effects on blebbing in these cells does not\nreflect a lack of Ca²⁺ requirement, but rather a divergence in how Ca²⁺ entry pathways are functionally\ndeployed.\nWe have incorporated these viability data into the revised manuscript and explicitly clarify that differential\nallocation of Ca²⁺ entry pathways to survival versus protrusive functions likely underlies the observed cell-\ntype–specific responses. The mechanistic basis for this functional partitioning remains an important topic\nfor future investigation.\nPoint-by-point responses to the reviewers' comments\nReviewer #1 (Remarks to the Author):\nI thank the authors for carefully addressing all my comments. I have one remaining concern relating\nthe calcium levels as imaged by Fluo4 in extended Figure 9. Why are the levels of calcium in the\nnucleus so high compared to the cytosol? That is not typical and may indicate highly stressed cells.\n- We thank the reviewer for this observation. AM-ester Ca²⁺ indicators such as Fluo-4 are known to\nexhibit intracellular compartmentalization after loading, which can lead to non-uniform fluorescence\ndistribution, including apparent nuclear enrichment. Consistent with this property, we observed similar\nnuclear accumulation of Fluo-4 in almost all cells across independent experiments using the same cell\ntype under different experimental conditions.\nExtended Data Fig. 9 shows representative images illustrating the spatial distribution of Fluo-4\nfluorescence. Importantly, quantitative analysis of Ca²⁺ signals was performed in the main figures,\nwhere nuclear regions were excluded from the measurements. Thus, the conclusions regarding Ca²⁺\nenrichment in bleb regions are based on comparisons between bleb areas and adjacent cytosolic\nregions while excluding the nucleus and are therefore not affected by nuclear fluorescence intensity.\nThis point has now been clarified in the revised manuscript. (Line 832-833).\nReviewer #2 (Remarks to the Author):\nThank you for the revised version. You have address all of my comments adequately.\n- We thank the reviewer for the careful evaluation of our manuscript and for the constructive comments\nprovided during the review process. We appreciate the reviewer’s positive assessment of the revised\nversion.\nReviewer #3 (Remarks to the Author):\nThe authors have addressed my concerns and comments by adding additional controls and impactful\ndata where required or by reanalyzing and drawing conclusions sticking the available data.\nOf note , the discussion has now an impactful meaning by\n- highlighting the technical limits of the present study and how it can be interpreted\n- the conceptual limits of the present framework\n- the future directions that are worthy of interest but out of the scope of the study\nI feel that the manuscript has now significantly improved and can be further processed for publication.\nAs a side note, I would like to apologize to the authors for the typo I missed in my comment 4. The\nword \"bread\" was meant to be \"bead\".\n- We sincerely thank the reviewer for the careful evaluation of our manuscript and for the constructive\ncomments that helped us improve the study. We are pleased that the reviewer finds the revised\nmanuscript significantly improved."}]

Decision letter