A novel tumor-immune microenvironment (TIME)-on-Chip mimics three dimensional neutrophil-tumor dynamics and neutrophil extracellular traps (NETs)-mediated collective tumor invasion
Open accessAbstract
Neutrophils are the most abundant type of leukocytes in the blood, traditionally regarded as the first immune responders to infections and inflammations. In the context of tumors, neutrophils have been shown to possess both tumor-promoting and tumor-limiting properties. A better understanding of the inter-cellular dynamics between the neutrophils and aggregated tumors could possibly shed light on the different modalities of neutrophil involvement in tumor progression. To study in-vitro the interactional dynamics of neutrophils and growing tumor aggregates, in this work, we engineered a novel, microfluidics-integrated, three-dimensional (3D) tumor-immune microenvironment (TIME)-on-Chip device, and we investigated the effect of neutrophils on the inception of collective 3D invasion of ovarian tumor cells. Herein, tumor spheroids generated and cultured on hydrogel based multi-microwell plates, and embedded within collagen matrix of defined thickness, were magnetically hybrid-integrated with a 3D bioprinting enabled microfluidic system fabricated on a porous membrane and carrying neutrophils. This setting recreated a typical TIME in-vitro to model dynamic neutrophil migration and 3D tumor invasion. Using this device, we observed that neutrophils respond to the growing tumor spheroids through both chemotaxis and generation of neutrophil extracellular traps (NETs). The formation of NETs stimulated the reciprocation of tumor cells from their aggregated state to collectively invade into the surrounding collagen matrix, in a manner more significant compared to their response to known tumor-derived stimulants such as transforming growth factor and Interleukin- 8. This effect was reversed by drug-induced inhibition of NETs formation, suggesting that induction of NETs by cancer cells could be a pro-migratory tumor behavior. Further, we additionally report a previously unidentified, location-dictated mechanism of NETosis, in which NETs formation within the stromal extracellular collagen matrix around the spheroids, and not tumor-contacted NETs, is important for the induction of collective invasion of the ovarian tumor cells, thus providing a rationale for new anti-tumor therapeutics research.
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Written from the paper’s full text.
TL;DR
The authors engineered a microfluidics-integrated 3D tumor-immune microenvironment (TIME)-on-Chip to model neutrophil–ovarian tumor spheroid interactions. They found that OVCAR-3 spheroids induce neutrophil chemotaxis and NETosis, and that stromal NETs—not tumor-contacted or vascular NETs—drive collective 3D tumor invasion, which is reversed by NET inhibition.
Key findings
- Neutrophils migrated toward OVCAR-3 spheroids by chemotaxis as early as 30 min after loading; maximum infiltration was ∼10% of all microchannel neutrophils, with ∼6% influx into the stromal collagen layer. Without spheroids, no random neutrophil migration into collagen was observed.
- Under flow (2 µl h⁻¹), neutrophils infiltrated significantly deeper into spheroids than under static conditions, with mean penetration ∼10% greater (p < 0.0001 for S1 and S2; p < 0.05 for S3). Spheroids closest to the microchannel recruited the most neutrophils.
- OVCAR-3 spheroids induced NET formation within 1 h, detected by Sytox and citrullinated histone H3. NETs stimulated collective 3D invasion more significantly than TGF-β or IL-8; 10 µM Sivelestat reduced inter-tumoral NET density (p < 0.0001) and spheroid distortion (p < 0.0001).
- Stromal NETs, not tumor-contacted NETs or vascular NETs, mediated collective invasion: neutrophils pipetted directly onto spheroids produced NETs but negligible distortion, and PMA-stimulated channel NETs (>60% NETosed) also produced negligible spheroid distortion. Maximum NET density in collagen did not exceed 12%.
Methods
- Fabricated a TIME-on-Chip with OVCAR-3 spheroids in polyacrylamide hydrogel microwells (500 µm inner diameter, 200 µm opening, 600 µm spacing), embedded in 1.5 mg ml⁻¹ type-I collagen, magnetically integrated with 3D-bioprinted microfluidic channels (500 µm × 500 µm × 8 mm) on a 3 µm porous track-etched membrane. Neutrophils were isolated from healthy donor peripheral blood.
- Assays included 6 h and 24 h neutrophil–tumor interactions under no-flow and flow (2 µl h⁻¹) conditions; NETs detected with Sytox Green and anti-H3Citrulline; NET inhibition with 10 µM Sivelestat; stimulation with 500 nM PMA, 100 ng ml⁻¹ TGF-β, or 100 ng ml⁻¹ IL-8. Imaging used inverted fluorescence microscopy and z-stacks; diffusion was validated with 10 kDa FITC-dextran and COMSOL finite element modeling.
- Data expressed as mean ± SEM; comparisons used Student’s t-test or two-tailed one-way ANOVA with Tukey post-hoc; p < 0.05 considered significant.
Limitations
- The paper states that it is difficult to identify a specific tumor-infiltrating neutrophil subgroup among the heterogeneous population, that data are insufficient to establish relay signaling for NETs, and that no single cytokine or NET-associated autocrine/paracrine factor was identified as responsible for the effects.
- The paper notes that mechanobiological modalities of NETs–stroma interactions remain understudied and require better understanding of the mechanical evolution of NETs in specific tumor states.
- Evident from the text: findings are limited to in vitro OVCAR-3 spheroids and healthy donor neutrophils, with no in vivo validation; no tumor cell infiltration across the porous membrane was observed even with NETs; assays were limited to 6–24 h; and maximum stromal NET density remained ≤12%.
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Claims the paper makes
0 of 61 cited sources checked · 0 supported · 0 flagged
- Findingown data
The platform enables 3D interstitial fluid transport around the spheroids to support long-term cultures.
“our initial results demonstrate the capability of the platform to enable 3D interstitial fluid transport around the spheroids to support long-term cultures (supported by supplementary figure 5S).”
Quote in the paperSubstantial claim - Findingown data
NET formation induces collective invasion of OVCAR-3 ovarian tumor cells from their aggregated state into the surrounding collagen matrix.
“The formation of NETs stimulated the reciprocation of tumor cells from their aggregated state to collectively invade into the surrounding collagen matrix”
- Also stated in: § Introduction · boosted
“The results established that NET formation induces collective invasion of OVCAR-3 cells into the collagen”
Quote in the paperSubstantial claim - Also stated in: § Introduction · boosted
- Findingown data
The NET-induced collective invasion is more significant than the tumor cell response to known tumor-derived stimulants TGF-β and IL-8.
“in a manner more significant compared to their response to known tumor-derived stimulants such as transforming growth factor and Interleukin-8.”
Quote in the paperSubstantial claim - Findingown data
NETs formation within the stromal extracellular collagen matrix around the spheroids, and not tumor-contacted NETs, is important for the induction of collective invasion of ovarian tumor cells.
“NETs formation within the stromal extracellular collagen matrix around the spheroids, and not tumor-contacted NETs, is important for the induction of collective invasion of the ovarian tumor cells”
- Also stated in: § Introduction · plain
“the NETs that are formed around the spheroids in the stromal extracellular collagen matrix, and not the tumor-contacted NETs, predominantly mediate the collective tumor invasion behavior”
Quote in the paperSubstantial claim - Also stated in: § Introduction · plain
- Findingown data
The 2D finite element model showed close agreement with the experimental diffusion behavior within a 3 h timespan.
“The results of the FEM showed a close agreement with the experiment within a 3 h timespan (figure 2(A)(iii)).”
Quote in the paperSubstantial claim- The authors state this as an assumption: In the finite element model, diffusion of solute into the collagen happens across the porous interfaces from the microfluidic channel, with negligible mass transport across a fully cured UV glue surrounding the channel.
- Findingown data
Drug-induced inhibition of NETs formation reverses the NET-induced collective invasion effect.
“This effect was reversed by drug-induced inhibition of NETs formation”
- Also stated in: § Introduction · plain
“this behavior is reversed by drug-induced abrogation of the NETs.”
Quote in the paperSubstantial claim - Also stated in: § Introduction · plain
- Findingown data
The magnetic hybrid integration ensures connectivity across the interfaces of the different layers of the system.
“Thus, the magnetic hybrid integration ensures connectivity across the interfaces of the different layers of the system”
Quote in the paperSubstantial claim - Findingown data
Neutrophils migrated to the OVCAR-3 spheroids through the collagen matrix.
“Neutrophils migrated to the spheroids through the collagen matrix (supplementary figure 8S(i)).”
Quote in the paperSubstantial claim - Findingown data
No significant difference was observed in the distortion of spheroids due to either direct stimulation with PMA or by stimulation of neutrophils in the channel with PMA.
“No significant difference was observed in the distortion of spheroids due to either direct stimulation with PMA or by stimulation of neutrophils in the channel with PMA (figure 6(iii)).”
Quote in the paperSubstantial claim - Findingown data
After 24 h, even though a substantial population of neutrophils produced NETs, the resultant spheroid distortion was surprisingly negligible.
“We observed that after 24 h, even though a substantial population of these neutrophils produced NETs, (supplementary figure 15S), the resultant spheroid distortion was surprisingly negligible.”
Quote in the paperSubstantial claim - Findingown data
Irrespective of the NET density or the proximity of these NETs to the spheroids, the resultant spheroid distortion observed after 24 h was negligible.
“irrespective of the NET density or the proximity of these NETs to the spheroids, the resultant spheroid distortion observed after 24 h was negligible.”
Quote in the paperSubstantial claim - Findingreasoning
Sytox-stained features in the collagen layer surrounding the spheroids were markedly more spread out than those formed inside the tumors.
“In general, the Sytox stained features that were observed in the collagen layer surrounding the spheroids were markedly more spread out than those formed inside the tumors (supplementary figure 10S).”
Quote in the paperNot a substantial claim - Findingown data
3D tumor spheroids were consistently generated using the described method.
“We were able to consistently generate 3D tumor spheroids using the method described in section 2.2 (supplementary figure 1S).”
Quote in the paperSubstantial claim - Findingown data
The structures of extruded features from PMA stimulation were similar to those formed by neutrophils migrating towards the spheroids.
“The structures of the extruded features from PMA stimulation were similar to those formed by the neutrophils migrating towards the spheroids (supplementary figure 11S)”
Quote in the paperSubstantial claim - Findingown data
The fraction of NETs present within the spheroid domain invading into the collagen was significantly lower than the NETs fraction present within the intact domain of the distorted spheroids.
“the fraction of NETs present within the spheroid domain invading into the collagen was significantly lower than the NETs fraction present within the intact domain of the distorted spheroids (figure 4(C)(ii)).”
Quote in the paperSubstantial claim - Findingown data
Upon stimulation, the final projected area of distorted OVCAR-3 spheroids was always larger than the initial spheroid area (φ > 0).
“Upon stimulation, the cells migrated into the collagen matrix, and the final projected area of the distorted spheroids was always larger than the initial spheroid area (ϕ > 0)”
Quote in the paperSubstantial claim - Findingown data
The inter-tumoral NETs density significantly reduced (p < 0.0001) upon treating the neutrophils with Sivelestat.
“the inter-tumoral NETs density significantly reduced (p < 0.0001) upon treating the neutrophils with Sivelestat (figure 4(B)(ii)).”
Quote in the paperSubstantial claim - Findingown data
The magnetically enabled hybrid integration approach provided robustness to maintain the integrated assembly free of distortion from vibrations associated with handling the device.
“In general, the magnetically enabled hybrid integration approach provided robustness to maintain the integrated assembly free of distortion from vibrations associated with handling the device”
Quote in the paperNot a substantial claim - Findingown data
In the absence of spheroids, no random migration of neutrophils into the collagen region was observed.
“in the absence of spheroids, no random migration of the neutrophils into the collagen region was observed (supplementary figure 9S)”
Quote in the paperSubstantial claim - Findingown data
The percentage of NET structures occupying the collagen layer increased proportionally to the initial number of neutrophils present in the channel.
“the percentage of NET structures occupying the collagen layer (calculated from z-stack images) increased proportionally to the initial number of neutrophils present in the channel.”
Quote in the paperSubstantial claim - Findingown data
The distortion with neutrophil infiltration was much more pronounced and evident than patterns observed following stimulation with TGF-β or IL-8.
“This distortion was much more pronounced and evident than the patterns observed following stimulation with TGF-β or IL-8”
Quote in the paperSubstantial claim - Findingown data
The NETs present in the invasive domain were not seen specifically at the leading edge of the invading cells.
“the NETs present in the invasive domain were not seen specifically at the leading edge of the invading cells”
Quote in the paperSubstantial claim - Findingown data
TGF-β or IL-8 stimulation significantly increased spheroid distortion (p < 0.001) compared to unstimulated spheroids.
“However, when exposed to the stimulants, TGF-β or IL-8, the resultant distortion of the spheroids was significantly higher (p < 0.001) than unstimulated spheroids (figures 2(B)(i) and B(ii)).”
Quote in the paperSubstantial claim - Findingown data
OVCAR-3 spheroids displayed negligible distortion when exposed to a rich, PMA-stimulated NETs microenvironment outside the collagen region.
“On the contrary, the OVCAR-3 spheroids displayed negligible distortion when exposed to a rich, PMA-stimulated NETs microenvironment outside the collagen region.”
Quote in the paperSubstantial claim - Findingown data
Whenever there was a higher availability of NETs within the stromal collagen region, the resultant spheroid distortion was also greater.
“whenever there was a higher availability of NETs within the stromal collagen region, the resultant spheroid distortion was also greater (figure 5(A)(iii)).”
Quote in the paperSubstantial claim - Findingown data
Different microfluidic channel configurations with controlled positioning with respect to the spheroids along all three coordinate axes were successfully fabricated and tested.
“We successfully fabricated and tested different microfluidic channel configurations with controlled positioning with respect to the spheroids along all three coordinate axes”
Quote in the paperSubstantial claim - Findingown data
At baseline, OVCAR-3 cells did not display spontaneous migration into the collagen matrix for at least 24 h.
“At baseline, OVCAR-3 cells did not display spontaneous migration into the collagen matrix for at least 24 h.”
Quote in the paperSubstantial claim - Findingown data
Mean E-cadherin expression of OVCAR-3 spheroids was significantly reduced under stimulated conditions.
“The mean E-cadherin expression of the spheroids was significantly reduced under stimulated conditions (figure 2(B)(iii)).”
Quote in the paperSubstantial claim - Findingown data
Our data do not support a role for NETs even within 'leaky vasculature'-like microfluidic channels in enhancing the extravasation of the tumor cells into the channel.
“However, our data do not support a role for NETs even within ‘leaky vasculature’-like microfluidic channels in enhancing the extravasation of the tumor cells into the channel.”
Quote in the paperSubstantial claim - Findingown data
Within 1 h, neutrophils extruded webs of DNA detectable by Sytox stain under both flow and no-flow conditions.
“Within 1 h, neutrophils extruded webs of DNA that could be detected by the Sytox stain. This was observed under both flow and no-flow conditions (figure 3(D)).”
Quote in the paperSubstantial claim - Findingown data
In the presence of neutrophil infiltration, significant spheroid distortion was observed in all spheroids over 24 h.
“In the presence of neutrophil infiltration, however, significant spheroid distortion was observed in all the spheroids over 24 h.”
Quote in the paperSubstantial claim - Findingown data
The resultant spheroid distortion dropped significantly (p < 0.0001) compared to untreated neutrophil infiltration.
“Importantly, the resultant spheroid distortion also dropped significantly (p < 0.0001) compared to untreated neutrophil infiltration (figure 4(C)(i)).”
Quote in the paperSubstantial claim - Findingown data
The inception of neutrophil chemotaxis occurred as quickly as 30 min after loading the cells into the channel.
“The inception of neutrophil chemotaxis occurred as quickly as 30 min after loading of the cells into the channel.”
Quote in the paperNot a substantial claim - Findingnone given
The device design proposed in this work enables a rapid, reproducible method to integrate functional 3D spheroids with tissue-engineered perfusable microfluidic channels to mimic the tumor-stromal-vascular organization.
“The device design proposed in this work enables a rapid, reproducible method to integrate functional 3D spheroids with tissue-engineered perfusable microfluidic channels to mimic the tumor-stromal-vascular organization”
Quote in the paperSubstantial claim - Findingnone given
Overall, our TIME-on-Chip device enables sophisticated recapitulation of certain critical in-vivo biological events in a physiologically relevant 3D microenvironment.
“Overall, our TIME-on-Chip device enables sophisticated recapitulation of certain critical in-vivo biological events in a physiologically-relevant 3D-microenvironment, making this an extremely valuable tool for translational research”
Quote in the paperSubstantial claim - Findingown data
Our TIME-on-Chip model is capable of recreating in-vivo-like neutrophil extravasation and NETosis functionalities within a tumor microenvironment.
“Our TIME-on-Chip model is capable of recreating in-vivo like neutrophil extravasation and NETosis functionalities within a tumor microenvironment”
Quote in the paperSubstantial claim - Findingown data
Our device enabled the biomimicry of certain exclusive NETs-associated functionalities and their reciprocal effects on tumor development, which are otherwise impossible to implement on conventional in-vitro assay platforms.
“As seen from the results of this work, our device easily enabled the biomimicry of certain exclusive NETs-associated functionalities and their reciprocal effects on tumor development, which are otherwise impossible to implement on conventional in-vitro assay platforms”
Quote in the paperSubstantial claim - Interpretationown data
The simple 2D computational model can predict the diffusion behavior required to modify the design configuration of the TIME-on-Chip.
“This serves as a validation for the capability of our simple 2D computational model to predict the diffusion behavior, as required to modify the design configuration of our TIME-on-Chip”
Quote in the paperSubstantial claim- The authors state this as an assumption: In the finite element model, diffusion of solute into the collagen happens across the porous interfaces from the microfluidic channel, with negligible mass transport across a fully cured UV glue surrounding the channel.
- Premisecitation
NETs have been directly associated with the initiation and induction of tumor invasion, aiding metastasis.
“recently, through increasing evidence, NETs have been directly associated with the initiation and induction of tumor invasion, aiding metastasis [20, 33–36].”
Quote in the paperSubstantial claim0 of 5 sources checked- cites [20] Lee, 2019, Neutrophils facilitate ovarian cancer premetastatic niche formation in the omentum, J. Exp. Med. source
Not checked in the free run.
- cites [33] Cools-Lartigue, 2013, Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis, J. Clin. Invest. source
Not checked in the free run.
- cites [34] Tohme, 2016, Neutrophil extracellular traps promote the development and progression of liver metastases after surgical stress, Cancer Res. source
Not checked in the free run.
- cites [35] Park, 2016, Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps, Sci. Transl. Med. source
Not checked in the free run.
- cites [36] Albrengues, 2018, Neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice, Science source
Not checked in the free run.
- Premisecitation
The microfluidic channel replicates an intact vascular structure surrounding the tumor that has a junction gap of ∼2 μm.
“The microfluidic channel replicates an intact vascular structure surrounding the tumor that have a junction gap of ∼2 µm [48].”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [48] Wang, 2018, Tumor-vasculature-on-a-chip for investigating nanoparticle extravasation and tumor accumulation, ACS Nano source
Not checked in the free run.
- Interpretationown data
Induction of NETs by cancer cells could be a pro-migratory tumor behavior.
“suggesting that induction of NETs by cancer cells could be a pro-migratory tumor behavior.”
Quote in the paperSubstantial claim - Interpretationreasoning
The device configuration allows in-vivo-like 3D biochemical correspondence between the tumor cells, stroma and the vasculature.
“This configuration allows in-vivo like 3D biochemical correspondence between the tumor cells, stroma and the vasculature.”
Quote in the paperSubstantial claim- Rests on an assumption: The collagen layer filled between the spheroids and the microfluidic channel mimics the tumor stroma.
- Premisecitation
Chemotherapy-induced neutropenia has predicted improved survival rates among cancer patients.
“chemotherapy induced neutropenia has also predicted improved survival rates among cancer patients [12–17]”
Quote in the paperNot a substantial claim0 of 6 sources checked- cites [12] Kishida, 2009, Chemotherapy-induced neutropenia as a prognostic factor in advanced non-small-cell lung cancer: results from Japan Multinational Trial Organization LC00-03, Br. J. Cancer source
Not checked in the free run.
- cites [13] Han, 2012, Prognostic value of chemotherapy-induced neutropenia in early-stage breast cancer, Breast Cancer Res. Treat. source
Not checked in the free run.
- cites [14] Tewari, 2014, Chemotherapy-induced neutropenia as a biomarker of survival in advanced ovarian carcinoma: an exploratory study of the gynecologic oncology group, Gynecol. Oncol. source
Not checked in the free run.
- cites [15] He, 2020, Early onset neutropenia: a useful predictor of chemosensitivity and favorable prognosis in patients with serous ovarian cancer, BMC Cancer source
Not checked in the free run.
- cites [16] Ma, 2016, Prognostic value of chemotherapy-induced neutropenia at the first cycle in invasive breast cancer, Medicine source
Not checked in the free run.
- cites [17] Kasi, 2018, Chemotherapy-induced neutropenia as a prognostic and predictive marker of outcomes in solid-tumor patients, Drugs source
Not checked in the free run.
- Interpretationreasoning
The TIME-on-Chip device design mimics the physiology of a typical TIME in a 3D setting.
“This device design mimics the physiology of a typical TIME in a 3D setting wherein, neutrophils from the microfluidic channel could migrate towards the tumor spheroids in 3D through the collagen matrix, leading to NETosis and 3D tumor invasion into the stroma.”
- Also stated in: § Abstract · plain
“This setting recreated a typical TIME in-vitro to model dynamic neutrophil migration and 3D tumor invasion.”
Quote in the paperSubstantial claim- Rests on an assumption: The collagen layer filled between the spheroids and the microfluidic channel mimics the tumor stroma.
- Also stated in: § Abstract · plain
- Interpretationown data
NETs do not necessarily trap the more motile OVCAR-3 phenotypes and drag them out of their intact domain to initiate the tumor invasion cascade.
“suggesting that NETs do not necessarily trap the more motile OVCAR-3 phenotypes and drag them out of their intact domain to initiate the tumor invasion cascade.”
Quote in the paperSubstantial claim - Interpretationown data
NETs play an important role in OVCAR-3 cells acquiring a pro-migratory/invasive phenotype.
“Thus, it is clear that NETs play an important role in OVCAR-3 cells acquiring a pro-migratory/invasive phenotype.”
Quote in the paperSubstantial claim - Interpretationown data
The migration of OVCAR-3 was dependent on the down-regulation of E-Cadherin.
“In general, the migration of OVCAR-3 was dependent on the down-regulation of E-Cadherin.”
Quote in the paperSubstantial claim - Interpretationown data
Stromal NETs, and not the tumor-contacted NETs or the vascular NETs, play a significant role in mediating the collective invasion of OVCAR-3 from an aggregated state.
“Thus, taken together, our data clearly suggest that stromal NETs, and not the tumor-contacted NETs or the vascular NETs, play a significant role in mediating the collective invasion of OVCAR-3 from an aggregated state.”
- Also stated in: § Discussion · hedged
“The demonstrations from our experimental tumor proliferation assays suggest that NETs that are formed in the surrounding stroma of a TIME, and not the NETs generated by the TINs, regulate the collective migration and invasion of cancer cell aggregates.”
Quote in the paperSubstantial claim - Also stated in: § Discussion · hedged
- Premisecitation
TGF-β and IL-8 have been reported to be secreted by activated neutrophils.
“Both of these have also been reported to be secreted by activated neutrophils [82, 83].”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [82] Grotendorst, 1989, Production of transforming growth factor beta by human peripheral blood monocytes and neutrophils, J. Cell. Physiol. source
Not checked in the free run.
- cites [83] Glowacka, 2010, IL-8, IL-12 and IL-10 cytokines generation by neutrophils, fibroblasts and neutrophils-fibroblasts interaction in psoriasis, Adv. Med. Sci. source
Not checked in the free run.
- Interpretationown data
Neutrophils respond to tumor spheroids through chemotaxis.
“thereby confirming that neutrophils respond to the tumor spheroids through chemotaxis.”
Quote in the paperSubstantial claim - Interpretationown data
OVCAR-3 cells induce neutrophils to NETose.
“thereby confirming that OVCAR-3 cells induce the neutrophils to NETose.”
Quote in the paperSubstantial claim - Interpretationown data
In a 3D setup, OVCAR-3 cells migrated and invaded as a group rather than as single cells.
“implying that in a 3D setup, OVCAR-3 cells migrated and invaded as a group rather than as single cells.”
- Also stated in: § Discussion · plain
“Our data shows that OVCAR-3 cells migrated cumulatively rather than as individual cells disseminating from the original aggregate.”
Quote in the paperSubstantial claim - Also stated in: § Discussion · plain
- Interpretationown data
NET-induced collective tumor invasion is specific to the interaction of the NETs with the collagen matrix surrounding the spheroids.
“NET-induced collective tumor invasion is specific to the interaction of the NETs with the collagen matrix surrounding the spheroids.”
- Also stated in: § Discussion · hedged
“Therefore, we believe that the collective invasion of tumor spheroids seen in our experiments is specific to the NETs-collagen interactions, and it is simply not due to the tumor-chemotactic factors or other components ejected out of the neutrophils during NETosis.”
Quote in the paperSubstantial claim - Also stated in: § Discussion · hedged
- Interpretationown data
Tumor cell-derived factors that stimulate neutrophil mobilization through chemotaxis do not necessarily also trigger NETosis.
“This also indicates that tumor cell-derived factors that stimulate neutrophil mobilization through chemotaxis do not necessarily also trigger NETosis.”
Quote in the paperNot a substantial claim - Interpretationcitation
Proteins typically associated with NET structures (elastase, cathepsin G, myeloperoxidase, and histones) could interact and bind directly with collagen and other ECM proteins to form complex interlinked structures that alter matrix architecture.
“the proteins [90] that are typically associated with the NET structures (elastase, cathepsin G, myeloperoxidase, and histones) could interact and bind directly with the collagen and other proteins present within the ECM, to form complex interlinked structures that result in altered properties of the matrix architecture”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [90] Castanheira, 2019, Neutrophils and NETS in modulating acute and chronic inflammation, Blood source
Not checked in the free run.
- Interpretationown data
Without external NET-inducing agents such as PMA, tumor-associated inflammatory factors by themselves trigger naive neutrophils to produce NETs.
“We also demonstrated that without external NET-inducing agents such as the PMA, tumor-associated inflammatory factors by themselves trigger naive neutrophils to produce NETs.”
Quote in the paperSubstantial claim - Premisecitation
Unstimulated OVCAR-3 cells could remain largely in a proliferation-quiescent state within a 3D matrix.
“it has been reported that unstimulated OVCAR-3 could remain largely in a proliferationquiescent state within a 3D matrix [64, 66].”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [64] Xu, 2014, Construction and characteristics of an E-cadherin-related three-dimensional suspension growth model of ovarian cancer, Sci. Rep. source
Not checked in the free run.
- cites [66] Hallas-Potts, 2019, Ovarian cancer cell lines derived from non-serous carcinomas migrate and invade more aggressively than those derived from high-grade serous carcinomas, Sci. Rep. source
Not checked in the free run.
- Interpretationown data
OVCAR-3 cells appeared to collectively invade into the collagen matrix in a highly directional manner.
“the OVCAR-3 cells appeared to collectively invade into the collagen matrix in a highly directional manner (top row in figure 4(A)).”
Quote in the paperSubstantial claim - Interpretationreasoning
Upon stimulation, OVCAR-3 spheroids collectively invaded the collagen matrix.
“Therefore, our results suggest that upon stimulation, the OVCAR-3 spheroids collectively invaded the collagen matrix.”
Quote in the paperSubstantial claim - Interpretationown data
The products expelled out of NETs were not directly mediating the spheroid distortion.
“Therefore, clearly, the products expelled out of NETs were not directly mediating the spheroid distortion.”
Quote in the paperSubstantial claim - Interpretationown data
Blocking NET formation impeded the neutrophils' ability to stimulate OVCAR-3 invasion into the collagen matrix.
“Thus, blocking NET formation impeded the neutrophils’ ability to stimulate OVCAR-3 invasion into the collagen matrix.”
Quote in the paperSubstantial claim - Interpretationreasoning
NETs could regulate the initiation of tumor invasion through biophysical tailoring of the tissues in and around a TIME.
“This observation further strengthens the argument that amidst other multifarious dynamics, NETs could regulate the initiation of tumor invasion through biophysical tailoring of the tissues in and around a TIME”
Quote in the paperSubstantial claim - Premisecitation
Non-destructive retrieval of cells or tissue samples from different micro-compartments of enclosed microfluidic Tumor-on-Chip systems, and simultaneous maintenance of compatibility with existing analysis and imaging systems for closer post-assay operations, can be extremely challenging.
“Consequently, non-destructive retrieval of the cells or tissue samples from the different micro-compartments of such systems, and simultaneous maintenance of compatibility with existing analysis and imaging systems for closer post-assay operations can be extremely challenging [105]”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [105] Sontheimer-Phelps, 2019, Modelling cancer in microfluidic human organs-on-chips, Nat. Rev. Cancer source
Not checked in the free run.
- Rests on an assumption: Most Tumor-on-Chip platforms with enclosed microfluidic channels are irreversibly bonded to render them leak-proof.
- Interpretationreasoning
Dismantling the architecture of NETs with DNAse treatment could reduce tumor invasion.
“Dismantling the architecture of the NETs with DNAse treatment could reduce tumor invasion”
Quote in the paperSubstantial claim- Rests on an assumption: DNAse treatment effectively degrades NETs and thus limits the complementary effects on tumor progression.
- Premisecitation
The degradation process of NETs leaves behind fragments of NETs that could still be biologically active.
“research shows that the degradation process leaves behind fragments of NETs which could still be biologically active [94]”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [94] Scozzi, 2019, Neutrophil extracellular trap fragments stimulate innate immune responses that prevent lung transplant tolerance, Am. J. Transplant. source
Not checked in the free run.
- May combine two claims. 25
- Classified as probably not a substantial claim. 31
May combine two claims.
Findingown dataNeutrophils respond to growing tumor spheroids through both chemotaxis and generation of neutrophil extracellular traps (NETs).
“Using this device, we observed that neutrophils respond to the growing tumor spheroids through both chemotaxis and generation of neutrophil extracellular traps (NETs).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataA maximum of ∼10% of all neutrophils from the microchannel infiltrated into the tumor, with ∼6% influx observed within the stromal collagen layer.
“overall, a maximum of ∼10% of all neutrophils from the microchannel infiltrated into the tumor, with ∼6% influx observed within the stromal collagen layer.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Findingnone givenThe PAAm formulation had an empirical shear modulus of ∼25 kPa.
“This PAAm formulation had an empirical shear modulus of ∼25 kPa.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataSpheroids closest to the microchannels recruited the maximum number of neutrophils, with the percentage recruitment dropping as the distance between spheroids and the channel increased.
“Spheroids closest to the microchannels recruited the maximum number of neutrophils, with the percentage recruitment dropping as the distance between spheroids and the channel increased (figure 3(C)).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Findingown dataMost of the spheroids appeared circular upon formation.
“Most of the spheroids appeared circular upon formation”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataThe average NET-induced spheroid polarization was significantly greater than those observed with TGF-β (p < 0.05) or IL-8 (p < 0.001).
“the average NET-induced spheroid polarization was significantly greater than those observed with TGF-β (p < 0.05) or IL-8 (p < 0.001).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe tumor aggregates were confined within the microwells, and subsequent addition of collagen on top, or magnetic attachment to the microfluidic channel, did not distort the spheroids.
“The tumor aggregates were confined within the microwells, and subsequent addition of collagen on top, or magnetic attachment to the microfluidic channel, did not distort the spheroids.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Findingown dataThe magnetic hybrid integration method enabled on-demand attach/detachment of the setup and provided maneuverability to precisely align the different sub-components under the microscope.
“This method of hybrid integration also enabled on-demand attach/detachment of the setup during both pre-and post-processing steps, and provided maneuverability to precisely align the different sub-components under the microscope.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataUnder flow, there was a significantly greater (p < 0.05) number of NET structures consistently present in each spheroid, with a significantly lower spread area.
“under flow, we found a significantly greater (p < 0.05) number of NETs structures consistently present in each of the spheroids (figure 3(E)(i)), albeit with a significantly lower spread area (figure 3(E)(ii)).”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
FindingreasoningThe versatility of the device facilitates rapid incorporation of additional biophysical functionalities of in-vivo relevance, such as interstitial fluidics or hemodynamics, without the need for drastic design modifications.
“The versatility of the device also facilitates rapid incorporation of additional biophysical functionalities of in-vivo relevance (for e.g. interstitial fluidics or hemodynamics) without the need for drastic design modifications”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Findingnone givenThe hybrid integration method proposed herein facilitates easy dismantling and reassembly of all sub-components for post-processing procedures such as immunostaining and imaging.
“Moreover, the hybrid integration method proposed herein facilitates easy dismantling and reassembly of all the sub-components for post-processing procedures such as immunostaining and imaging”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based).
Findingnone givenOur Tumor Immune Microenvironment-On-Chip model is simple to fabricate.
“Our Tumor Immune Microenvironment-On-Chip model is simple to fabricate and could be implemented in several potential applications in a broad range of scientific research”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Findingown dataHybrid integration with magnetic attachment can be implemented on a variety of macro-culture vessels, including multiwell plates and Transwell inserts.
“This hybrid integration with magnetic attachment can be implemented on a variety of macro-culture vessels, including multiwell plates and Transwell inserts (supplementary figure 17S)”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationHigh neutrophil numbers have generally been associated with poor prognosis and drug resistance in patients.
“their high numbers have, in general, been associated with poor prognosis and drug resistance in patients [9–11].”
Quote in the paperNot a substantial claim0 of 3 sources checked- cites [9] Schiffmann, 2019, Tumour-infiltrating neutrophils counteract anti-VEGF therapy in metastatic colorectal cancer, Br. J. Cancer source
Not checked in the free run.
- cites [10] Wang, 2018, Neutrophils infiltrating pancreatic ductal adenocarcinoma indicate higher malignancy and worse prognosis, Biochem. Biophys. Res. Commun. source
Not checked in the free run.
- cites [11] Carus, 2013, Tumour-associated CD66b+ neutrophil count is an independent prognostic factor for recurrence in localised cervical cancer, Br. J. Cancer source
Not checked in the free run.
Probably not a substantial claim: may not pass S4 (based).
Premisenone givenConventional 2D culture platforms cannot replicate certain functionalities of a TIME that are specific to three-dimensionality, such as neutrophil infiltration into a 3D tumor cluster, or the collective 3D invasion behavior of tumor cells.
“conventional two-dimensional (2D) culture platforms cannot replicate certain functionalities of a TIME that are specific to three-dimensionality, such as neutrophil infiltration into a 3D tumor cluster, or the collective 3D invasion behavior of the tumor cells.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationThe NETs-mediated tumor metastasis model has been clearly established for disseminating ovarian tumor cells.
“Whereas the NETs-mediated tumor metastasis model has been clearly established for disseminating ovarian tumor cells [20]”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [20] Lee, 2019, Neutrophils facilitate ovarian cancer premetastatic niche formation in the omentum, J. Exp. Med. source
Not checked in the free run.
Classified as probably not a substantial claim.
PremisecitationResearch has produced conflicting data on whether neutrophils promote the metastatic cascade or support immune reactions inhibiting tumor growth.
“research has produced conflicting data in explaining whether neutrophils could promote the metastatic cascade [18–22], or support immune reactions inhibiting tumor growth [23–26].”
Quote in the paperSubstantial claim0 of 9 sources checked- cites [18] Mayer, 2016, Neutrophil granulocytes in ovarian cancer-induction of epithelial-to-mesenchymal-transition and tumor cell migration, J. Cancer source
Not checked in the free run.
- cites [19] Singel, 2019, Mature neutrophils suppress T cell immunity in ovarian cancer microenvironment, JCI Insight source
Not checked in the free run.
- cites [20] Lee, 2019, Neutrophils facilitate ovarian cancer premetastatic niche formation in the omentum, J. Exp. Med. source
Not checked in the free run.
- cites [21] Yu, 2017, TNFα-activated mesenchymal stromal cells promote breast cancer metastasis by recruiting CXCR2+ neutrophils, Oncogene source
Not checked in the free run.
- cites [22] Wang, 2019, Tumor-contacted neutrophils promote metastasis by a CD90-TIMP-1 juxtacrine–paracrine loop, Clin. Cancer Res. source
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- cites [23] Yoshida, 2018, Intraperitoneal neutrophils activated by KRAS-induced ovarian cancer exert antitumor effects by modulating adaptive immunity, Int. J. Oncol. source
Not checked in the free run.
- cites [24] Muqaku, 2020, Neutrophil extracellular trap formation correlates with favorable overall survival in high grade ovarian cancer, Cancers source
Not checked in the free run.
- cites [25] Lee, 2000, IL-8 reduced tumorigenicity of human ovarian cancer in vivo due to neutrophil infiltration, J. Immunol. source
Not checked in the free run.
- cites [26] Liu, 2020, LPS and IL-8 activated umbilical cord blood-derived neutrophils inhibit the progression of ovarian cancer, J. Cancer source
Not checked in the free run.
May combine two claims.
Study design and conductcitationThe neutrophil isolation method depletes all other cells from blood, leaving the neutrophils unstimulated.
“This neutrophil isolation method depletes all other cells from blood, leaving the neutrophils unstimulated.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [53] Chandrasekaran, 2017, Temporal gradients limit the accumulation of neutrophils toward sources of chemoattractant, Microsyst. Nanoeng. source
Not checked in the free run.
May combine two claims.
PremisecitationNeutrophils can respond to tumor-derived chemotactic stimuli by extravasating from blood vessels into tumor tissue through chemotaxis, and migrate towards growing tumor aggregates within the stroma, releasing NETs along the way.
“neutrophils could respond to the tumor-derived chemotactic stimuli by extravasating from the blood vessels into the tumor tissue through chemotaxis [44], and migrate towards the growing tumor aggregates within the stroma, releasing NETs along the way [45]”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [44] Tazzyman, 2011, Inhibition of neutrophil infiltration into A549 lung tumors in vitro in vivo using a CXCR2-specific antagonist is associated with reduced tumor growth, Int. J. Cancer source
Not checked in the free run.
- cites [45] Teijeira, 2020, Cxcr1 and cxcr2 chemokine receptor agonists produced by tumors induce neutrophil extracellular traps that interfere with immune cytotoxicity, Immunity source
Not checked in the free run.
Classified as probably not a substantial claim. May combine two claims.
PremisecitationCollectively migrating tumor clusters have greater invasive capacity and higher resistance to therapeutic treatments than individually migrating tumor cells.
“The collectively migrating tumor clusters have been detected even in the early stage of cancers [43], and are said to have greater invasive capacity and higher resistance to the therapeutic treatments than the individually migrating tumor cells.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [43] Yang, 2019, An emerging tumor invasion mechanism about the collective cell migration, Am. J. Transl. Res.
Not checked in the free run.
May combine two claims.
InterpretationreasoningThe device design facilitates replicating neutrophil infiltration into the tumor tissue across the porous membrane and enables the study of tumor invasion.
“This device design not only facilitates the feasibility of replicating neutrophil infiltration into the tumor tissue across the porous membrane, but also enables the study of tumor invasion.”
Quote in the paperSubstantial claimClassified as probably not a substantial claim.
PremisecitationNeutrophils constitute a significant portion of the immune cells present within a TIME.
“Neutrophils constitute a significant portion of the immune cells present within a TIME [4–8]”
Quote in the paperSubstantial claim0 of 5 sources checked- cites [4] Powell, 2016, Neutrophils in the tumor microenvironment, Trends Immunol. source
Not checked in the free run.
- cites [5] Kitano, 2018, Tumour-infiltrating inflammatory and immune cells in patients with extrahepatic cholangiocarcinoma, Br. J. Cancer source
Not checked in the free run.
- cites [6] Hiramatsu, 2018, Neutrophils in primary gastric tumors are correlated with neutrophil infiltration in tumor-draining lymph nodes and the systemic inflammatory response, BMC Immunol. source
Not checked in the free run.
- cites [7] Yin, 2019, Prognostic significance of neutrophil–lymphocyte ratio (NLR) in patients with ovarian cancer: a systematic review and meta-analysis, Medicine source
Not checked in the free run.
- cites [8] Swierczak, 2015, Neutrophils: important contributors to tumor progression and metastasis, Cancer Metastasis Rev. source
Not checked in the free run.
May combine two claims.
Interpretationown dataThe TIME-on-Chip can recreate and analyze the locomotion behavior of 3D tumor cultures and carry out integrated labeling and post-assay analyses directly on chip.
“Overall, these results validate the utility of our TIME-on-Chip to recreate and analyze the locomotion behavior of 3D tumor cultures, and carry out integrated labeling and post-assay analyses directly on chip.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataOVCAR-3 spheroids induce neutrophil response through both chemotaxis and NETosis.
“Altogether, our results clearly establish that OVCAR-3 spheroids induce neutrophil response through both chemotaxis and NETosis.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S4 (based). May combine two claims.
Study design and conductnone givenDatasets were normally distributed, with similar variances between compared groups.
“Datasets were normally distributed, with similar variances between compared groups.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataNETs stimulate the invasion of OVCAR-3 cells into the collagen matrix and inhibiting NET formation abrogates tumor invasion.
“NETs stimulate the invasion of OVCAR-3 cells into the collagen matrix and inhibiting NET formation abrogates tumor invasion.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationDepletion of a few tumor-derived factors such as IL-8, G-CSF, GROα, or GROβ could diminish NET formation.
“previous works have shown that depletion of a few tumor-derived factors [20] such as IL-8, G-CSF, GROα, or GROβ could diminish NET formation.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [20] Lee, 2019, Neutrophils facilitate ovarian cancer premetastatic niche formation in the omentum, J. Exp. Med. source
Not checked in the free run.
Classified as probably not a substantial claim. May combine two claims.
InterpretationcitationPhysical parameters of the collagen matrix such as stiffness, pore size, and fiber re-organization could differ due to NETs, leading to modified chemical cues, adhesive ligand densities, or availability of binding sites to integrins and other matrix receptors.
“Consequently, it is possible that the physical parameters of the collagen matrix such as stiffness, pore size, and fiber re-organization could differ [91], leading to modified chemical cues, adhesive ligand densities, or the availability of binding sites to integrins and other matrix receptors”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [91] Velez, 2017, 3D collagen architecture induces a conserved migratory and transcriptional response linked to vasculogenic mimicry, Nat. Commun. source
Not checked in the free run.
May combine two claims.
Interpretationown dataIn this work, neutrophils respond to a growing tumor aggregate through two mechanisms, namely chemotaxis and NETosis, and the interaction with neutrophils also elicited a complementary response from the tumor cells through migration and invasion into the stroma.
“In this work, we demonstrated that neutrophils respond to a growing tumor aggregate through two mechanisms, namely chemotaxis and NETosis, and further we found that the interaction with neutrophils also elicited a complementary response from the tumor cells through migration and invasion into the stroma.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationIntravascular NETs can increase local vascular permeability, which would permit cancer cells to extravasate more easily.
“It has been reported that intravascular NETs can increase local vascular permeability [87, 88], which would permit cancer cells to extravasate more easily.”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [87] Kolaczkowska, 2015, Molecular mechanisms of NET formation and degradation revealed by intravital imaging in the liver vasculature, Nat. Commun. source
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- cites [88] Strilic, 2017, Intravascular survival and extravasation of tumor cells, Cancer Cell source
Not checked in the free run.
May combine two claims.
Interpretationown dataFluid flow around neutrophils activates them differently and could affect their response to tumors.
“These results suggest that fluid flow around the neutrophils activate them differently, and could affect their response to tumors.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
InterpretationreasoningThe regulation of collective OVCAR-3 migration by stromal neutrophil extracellular traps is possibly mediated through biophysical transduction.
“it is possible that the regulation of collective OVCAR-3 migration by the stromal NETs is mediated through biophysical transduction”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
PremisecitationStromal-tumor interactions are highly dependent on the physical properties of the extracellular matrix.
“Stromal-tumor interactions are highly dependent on the physical properties of the ECM [89]”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [89] Poltavets, 2018, The role of the extracellular matrix and its molecular and cellular regulators in cancer cell plasticity, Front. Oncol. source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationOVCAR-3 cells are generally known to form loose aggregates.
“though OVCAR-3 cells are generally known to form loose aggregates [54].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [54] Heredia-Soto, 2018, High-throughput 3-dimensional culture of epithelial ovarian cancer cells as preclinical model of disease, Oncotarget source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenComparative data analysis of populations was performed without pre-specifying a required effect size.
“In general, comparative data analysis of populations was performed without pre-specifying a required effect size.”
Quote in the paperNot a substantial claimMay combine two claims.
PremisecitationPMA could have counter-effects upon direct interaction with tumors, which may lead to spurious results in a co-culture system.
“PMA could also have counter-effects upon direct interaction with tumors [84, 85], which may lead to spurious results in a co-culture system.”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [84] He, 2010, Phorbol ester phorbol-12-myristate-13-acetate induces epithelial to mesenchymal transition in human prostate cancer ARCaPE cells, Prostate source
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- cites [85] Wong, 2016, Phorbol myristate acetate suppresses breast cancer cell growth via down-regulation of P-Rex1 expression, Protein Cell source
Not checked in the free run.
Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing). May combine two claims.
InterpretationreasoningThe success of Tumor-on-Chips depends not only on the novelty of their applications but also on their ease of implementation.
“Thus, the success of Tumor-on-Chips not only depends on the novelty of their applications, but also in their ease of implementation”
Quote in the paperNot a substantial claim- Rests on an assumption: Most Tumor-on-Chip platforms with enclosed microfluidic channels are irreversibly bonded to render them leak-proof.
- Rests on an assumption: Tumor-on-Chips are more heterogeneous than conventional 2D cultures with more complex analysis parameters.
Probably not a substantial claim: may not pass S2 (checkable).
InterpretationreasoningOur work supports the necessity of studying tumors on a 3D platform that offers unique insights into tumor behavior compared with regular 2D cultures.
“Lastly, our work emphasizes the necessity for studying tumors on a 3D platform that offer unique insights into tumor behavior than regular 2D cultures”
Quote in the paperNot a substantial claimMay combine two claims.
PremisecitationConventional laboratory-based cancer models built on planar, 2D culture vessels could be too simplified to faithfully mimic the tissue-specific diversities of neutrophil dynamics, and thus may not accurately represent the pathophysiology of tumor progression within a TIME.
“conventional laboratory-based cancer models built on planar, 2D culture vessels could be way too simplified [96, 97] to faithfully mimic the tissue-specific diversities of neutrophil dynamics, and thus may not accurately represent the pathophysiology of tumor progression within a TIME”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [96] Eglen, 2015, Drug discovery goes three-dimensional: goodbye to flat high-throughput screening?, ASSAY Drug Dev. Technol. source
Not checked in the free run.
- cites [97] Santo, 2017, Drug screening in 3D in vitro tumor models: overcoming current pitfalls of efficacy read‐outs, Biotechnol. J. source
Not checked in the free run.
Probably not a substantial claim: may not pass S2 (checkable).
InterpretationreasoningIncreasing the complexity of in-vitro tumor models may be a successful strategy only if the crucial components of a TIME are identified, replicated, and appropriately controlled for.
“However, increasing the complexity of in-vitro tumor models may be a successful strategy only if the crucial components of a TIME are identified, replicated, and appropriately controlled for”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
InterpretationreasoningThese NET-induced matrix features could potentially impact tumor motility and gene expression.
“Each of these features could potentially impact tumor motility and gene expression”
Quote in the paperSubstantial claimClassified as probably not a substantial claim.
PremisecitationTissue-engineered 3D tumor models consisting of spheroids or organoids grown within synthetic ECM constructs have enabled more rigorous tissue-level studies of the TIME and restore important architectural and stromal cues.
“Tissue-engineered 3D tumor models [100, 101], consisting of spheroids or organoids grown within synthetic ECM constructs, have enabled more rigorous tissue level studies of the TIME, and restore important architectural and stromal cues [102]”
Quote in the paperSubstantial claim0 of 3 sources checked- cites [100] Thoma, 2014, 3D cell culture systems modeling tumor growth determinants in cancer target discovery, Adv. Drug. Deliv. Rev. source
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- cites [101] Singh, 2020, Three-dimensional models of breast cancer–fibroblasts interactions, Exp. Biol. Med. source
Not checked in the free run.
- cites [102] Rizvi, 2013, Flow induces epithelial-mesenchymal transition, cellular heterogeneity and biomarker modulation in 3D ovarian cancer nodules, Proc. Natl Acad. Sci. source
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Probably not a substantial claim: may not pass S2 (checkable).
InterpretationreasoningThe results of our work underline the importance of incorporating three-dimensionality into the tumor microenvironment as a critical factor for gaining unique insights into significant biological events related to tumor growth and development.
“The results of our work underline the importance of incorporating three-dimensionality to Tumor Microenvironment as a critical factor for gaining unique insights into significant biological events related to tumor growth and development”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationSpheroids are generally considered similar to an early vascular stage of tumors.
“Spheroids [98, 99] are, in general, considered similar to an early vascular stage of tumors”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [98] Costa, 2016, 3D tumor spheroids: an overview on the tools and techniques used for their analysis, Biotechnol. Adv. source
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- cites [99] Nath, 2016, Three-dimensional culture systems in cancer research: focus on tumor spheroid model, Pharmacol. Ther. source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationDimensionality-based functional differences could also exist in neutrophil response.
“While dimensionality based functional differences could also exist in neutrophil response [95]”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [95] Yamahashi, 2015, Integrin associated proteins differentially regulate neutrophil polarity and directed migration in 2D and 3D, Biomed. Microdevices source
Not checked in the free run.
Probably not a substantial claim: may not pass S2 (checkable), S4 (based). May combine two claims.
Interpretationnone givenEngineering the dynamics of a TIME necessitates incorporation of multiple tissues, active vascular conduits, accountability of cell/tumor-specific differences in 3D spheroid formation, and arrangement of these ensembles in an intricate, biochemically relevant tumor-specific microenvironment without affecting sub-component functionalities.
“Engineering the dynamics of a TIME necessitates the incorporation of multiple tissues, inclusion of active vascular conduits, accountability of cell/tumor-specific differences in the ability to form 3D spheroids, and arrangement of each of these ensembles in an intricate and biochemically-relevant tumor-specific microenvironment without affecting the functionalities of each of the sub-components”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
InterpretationreasoningTumor-on-Chips are more heterogeneous than conventional 2D cultures with more complex analysis parameters, and could therefore require higher-resolution probing of cells or tissue components for confirmation of functional readouts.
“Tumor-on-Chips are more heterogeneous than the conventional 2D cultures with more complex analysis parameters, and could therefore require higher resolution probing of the cells or tissue components for the confirmation of functional readouts”
Quote in the paperNot a substantial claim- Rests on an assumption: Tumor-on-Chips are more heterogeneous than conventional 2D cultures with more complex analysis parameters.
May combine two claims.
PremisecitationSoft lithography and conventional microfluidics-based Tumor-on-Chips suffer tremendously from the limitation of ease of implementation, compounded by expensive clean-rooms for microfabrication processes that are not readily accessible to non-technical personnel.
“Soft lithography and conventional microfluidics-based Tumor-on-Chips suffer tremendously from this limitation, further compounded by the involvement of expensive clean-rooms for the microfabrication processes that are not readily accessible to non-technical personnel [104]”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [104] Chandrasekaran, 2017, Thermal scribing to prototype plastic microfluidic devices, applied to study the formation of neutrophil extracellular traps, Lab Chip source
Not checked in the free run.
May combine two claims.
PremisecitationNETs could stimulate ECM remodeling and promote integrin-mediated NETs-tumor interactions.
“NETs could stimulate ECM remodeling [36, 92] and to promote integrin-mediated NETs-tumor interactions [92, 93]”
Quote in the paperSubstantial claim0 of 3 sources checked- cites [36] Albrengues, 2018, Neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice, Science source
Not checked in the free run.
- cites [92] Kai, 2019, The extracellular matrix modulates the metastatic journey, Dev. Cell source
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- cites [93] Najmeh, 2017, Neutrophil extracellular traps sequester circulating tumor cells via β1-integrin mediated interactions, Int. J. Cancer source
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The paper relies on this without support.
Limited information is available about the interactions between NETs and aggregated tumor clusters that could explain possible downstream modalities of metastatic aggression of collectively invading tumors.
“limited information is available about the interactions between the NETs and aggregated tumor clusters that could explain the possible downstream modalities of metastatic aggression of collectively invading tumors.”
The authors state this as an assumption.
In the finite element model, diffusion of solute into the collagen happens across the porous interfaces from the microfluidic channel, with negligible mass transport across a fully cured UV glue surrounding the channel.
“The model was built based on the assumption that the diffusion of solute into the collagen happens across the porous interfaces from the microfluidic channel, with negligible mass transport across a fully cured UV glue surrounding the channel.”
The paper relies on this without support.
The collagen layer filled between the spheroids and the microfluidic channel mimics the tumor stroma.
“Collagen layer filled in between the spheroids and the channel mimics the tumor stroma.”
The paper relies on this without support.
The cues from a tumor microenvironment that induce neutrophils to form NETs are poorly understood.
“Till date, the cues from a Tumor Microenvironment (TME) that induce neutrophils to form NETs are poorly understood.”
The paper relies on this without support.
NETs play an active role in tumor metastasis.
“NETs are known to play an active role in tumor metastasis.”
The paper relies on this without support.
The dynamics of NET induction and the mechanisms by which NETs facilitate tumor metastasis are unclear.
“However, the dynamics of NET induction and the mechanisms by which NETs facilitate tumor metastasis are unclear.”
The paper relies on this without support.
The availability of NETs at the destination site is reflective of the metastatic potential of certain tumors.
“The availability of NETs at the destination site is also reflective of the metastatic potential of certain tumors.”
The paper relies on this without support.
Most in-vitro assays stimulate neutrophils with PMA for the production of NETs.
“Most in-vitro assays stimulate neutrophils with PMA for the production of NETs.”
The paper relies on this without support.
Not much attention has been paid to the mechanobiological modalities through which NETs accumulating around a tumor could influence the stroma and subsequently influence tumor development.
“However, till date, not much attention has been paid to the mechanobiological modalities through which the NETs accumulating around a tumor, could influence the stroma and subsequently influence tumor development”
The paper relies on this without support.
Most Tumor-on-Chip platforms with enclosed microfluidic channels are irreversibly bonded to render them leak-proof.
“Most of the Tumor-on-Chip platforms with enclosed microfluidic channels are irreversibly bonded to render them leak-proof”
The paper relies on this without support.
Tumor-on-Chips are more heterogeneous than conventional 2D cultures with more complex analysis parameters.
“Tumor-on-Chips are more heterogeneous than the conventional 2D cultures with more complex analysis parameters”
The paper relies on this without support.
DNAse treatment effectively degrades NETs and thus limits the complementary effects on tumor progression.
“While DNAse treatment effectively degrades the NETs and thus limits the complementary effects on tumor progression”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Microfluidic models have the capability to thoroughly model the tumor immune microenvironment comprised of endothelial, stromal and immune cell populations, making them useful preclinical tools to enhance our understanding of basic tumor pathology, immune cell exclusion and immunosuppression.
“Likewise, microfluidic models have the capability to thoroughly model the tumor immune microenvironment comprised of endothelial, stromal and immune cell populations (Miller et al., 2020; Surendran et al., 2021; Hachey et al., 2023a); making them useful preclinical tools to enhance our understanding of basic tumor pathology, immune cell exclusion and immunosuppression.”
From Microphysiological systems as models for immunologically ‘cold’ tumors (2024) · cites this paper as Surendran et al., 2021
Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.
Experimental — accuracy not yet measuredNET formation stimulated elongation of OVCAR-3 cells, induced loss of circularity of tumor spheroids, enhanced tumor migratory behavior, and induced tumor cell collective invasion into the collagen matrix in a highly directional manner.
“Subsequently, NET formation stimulated the elongation of OVCAR-3 cells, induced loss of circularity of tumor spheroids, enhanced tumor migratory behavior and induced tumor cell collective invasion into the collagen matrix in a highly directional manner.”
From Microphysiological systems as models for immunologically ‘cold’ tumors (2024) · cites this paper as Surendran et al., 2021
The Results section, “NETs stimulate OVCAR-3 invasion” (page unavailable), reports loss of spheroid circularity and marginal elongation at parts of the spheroid periphery, but not elongation of individual OVCAR-3 cells. It also reports NET-associated spheroid distortion, enhanced invasion, and highly directional collective invasion into collagen. The loss of circularity correlated with both NETs and intact neutrophils, so the passage does not establish that NET formation alone caused it. (LLM, conf=0.86)
Experimental — accuracy not yet measuredLess than a handful of ovarian cancer-based microfluidic chips incorporate immune cells, while the TME plays an essential role in tumor initiation, development, and therapy response.
“Less than a handful of ovarian cancer-based micro-fluidic chips incorporates immune cells, however, the TME plays an essential role in tumor initiation, development and therapy response (Surendran et al., 2021; Bouquerel et al., 2023).”
From Microphysiological systems as models for immunologically ‘cold’ tumors (2024) · cites this paper as Surendran et al., 2021
The Introduction says the tumor-immune microenvironment influences tumor initiation, development, and response to therapies. The Abstract describes this paper’s ovarian tumor chip incorporating neutrophils, but the paper does not establish that fewer than a handful of ovarian cancer microfluidic chips incorporate immune cells. (LLM, conf=0.85)
Experimental — accuracy not yet measuredA perfusable and well-connected microvascular network was formed via self-organization of all cell types.
“which showed that a perfusable and well‐connected microvascular network (µVN) was formed via the self‐organization of all cell types.”
From 3D Biomimetic Models to Reconstitute Tumor Microenvironment In Vitro: Spheroids, Organoids, and Tumor‐on‐a‐Chip (2023) · cites this paper as [152]
The Discussion (page not available) describes perfusable microfluidic channels integrated with tumor spheroids. The Results section on dynamic 3D tumor cultures (page not available) says the channels were fabricated and positioned to resemble vasculature-like networks; it does not report a well-connected microvascular network formed by self-organization of all cell types. (LLM, conf=0.87)
Experimental — accuracy not yet measuredSeveral other tumor chips have been developed, encompassing features such as tumor vasculature, stromal cells, or immune cells, and offering advantages in terms of rapid fabrication, reproducibility, real-time imaging and analysis, capturing tumor type and heterogeneity, facilitating robust drug testing, target discovery and personalized therapy screenings, and enabling multicellular crosstalk.
“Besides this model, several other tumor chips have been developed, encompassing features such as tumor vasculature (Yu et al. (2019); Wan et al. (2023); Nashimoto et al. (2020); Neufeld et al. (2021); Kim et al. (2022); Mannino et al. (2017); Shirure et al. (2018), stromal cells (Yu et al. (2019); Ibrahim et al. (2022); Karakas et al. (2017), or immune cells (Surendran et al. (2021); Aung et al. (”
From Microphysiological systems as models for immunologically ‘cold’ tumors (2024) · cites this paper as Surendran et al., 2021
This process can be reversed via drug-induced inhibition of NET formation, potentially opening a new therapeutic avenue for limiting ovarian cancer metastasis in patients.
“Notably, this process can be reversed via the drug-induced inhibition of NETs formation, potentially opening a new therapeutic avenue for limiting ovarian cancer metastasis in patients.”
From Microphysiological systems as models for immunologically ‘cold’ tumors (2024) · cites this paper as Surendran et al., 2021
Upon introduction of neutrophils into the microfluidic channel, cells migrated through the collagen matrix towards ovarian spheroids and elicited in vivo-like neutrophil responses, such as tumor-mediated chemotaxis and NET formation.
“Interestingly, upon the introduction of neutrophils into the microfluidic channel, the cells migrated through the collagen matrix towards the ovarian spheroids and were able to elicit in vivo-like neutrophil responses, such as tumor-mediated chemotaxis and NET formation.”
From Microphysiological systems as models for immunologically ‘cold’ tumors (2024) · cites this paper as Surendran et al., 2021
Direct interaction of the μVN with human neural cells rendered the microvasculature with low permeability, comparable to that present in rat brains.
“Moreover, direct interaction of the µVN with human neural cells has rendered the microvasculature with low permeability, comparable to that present in rat brains.”
From 3D Biomimetic Models to Reconstitute Tumor Microenvironment In Vitro: Spheroids, Organoids, and Tumor‐on‐a‐Chip (2023) · cites this paper as [152]
The Results section on TIME-on-Chip describes OVCAR-3 tumor spheroids, neutrophils, and microfluidic channels, not a microvascular network interacting with human neural cells. The Discussion mentions ‘leaky vasculature’-like channels but gives no low-permeability measurement or comparison with rat brains. Searches for neural cells, microvascular networks, and rat brains found no passage addressing the claim. (LLM, conf=0.76)
Experimental — accuracy not yet measured
The citation lists haven’t been fetched yet
References 113 · Cited by 99
- Authors
- Surendran, V., Rutledge, D., Colmon, R., & Chandrasekaran, A.
- Affiliations
- North Carolina Agricultural and Technical State University (US)
- Venue
- Biofabrication
- Publisher
- IOP Publishing
- Published
- 2021-04-08
- Volume
- 13
- Issue
- 3
- Pages
- 035029
- ISSN
- 1758-5082 (ISSN-L)
- 1758-5090
- PMC
- PMC8990531
- OpenAlex
- W3127400878
- Semantic Scholar
- fe1e61ba91b37f56c969190378cb9d19ce91de1c
- Cited by
- 99
- References
- 113
- Citations by year
- 2026: 13 · 2025: 21 · 2024: 21 · 2023: 19 · 2022: 19 · 2021: 6
- FWCI
- 4.94
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Biofabrication (publishedVersion, cc-by)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion, cc-by-nc-nd)
- Funders
- College of Engineering , NC A&T State University (Start-up Funds)
- National Institutes of Health (SC2-GM136523)
- Topics
- Physical Sciences › Engineering › Biomedical Engineering › 3D Printing in Biomedical Research
- Life Sciences › Immunology and Microbiology › Immunology › Neutrophil, Myeloperoxidase and Oxidative Mechanisms
- Life Sciences › Immunology and Microbiology › Immunology › Immune cells in cancer
- Keywords
- tumor immune microenvironment, neutrophils, neutrophil extracellular traps, NETosis, ovarian tumor cells, tumor spheroids, microfluidics, 3D bioprinting, collagen matrix, tumor stroma, chemotaxis
- MeSH terms
- Cell Movement, Humans, Neoplasms, Neutrophils, Tumor Microenvironment, Extracellular Traps
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-07.
Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-07.
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