Journal: Brazilian Journal of Medical and Biological Research

Article Title: LTF regulates glioblastoma progression and temozolomide resistance via the NF-κB signaling pathway

doi: 10.1590/1414-431X2025e13821

Figure Lengend Snippet: Lactotransferrin (LTF) competitively bound p65. A , Phosphorylation levels of p65 in control or LTF-depleted glioblastoma (GBM) cells was analyzed by western blot. B , U251 cells were transfected with LTF with or without four phosphatases. Phosphorylation levels of p65 in control or LTF-depleted GBM cells were analyzed by western blot. C , Immunoprecipitates were prepared with anti-LTF antibody. p65 and PP2A expression in immunoprecipitates was measured by western blot. D , Control and LTF-depleted cells were transfected with Flag-p65. Immunoprecipitates were prepared with anti-Flag antibody. PP2A expression in immunoprecipitates was measured by western blot. E , Schematic representation of Flag-tagged full-length LTF (#1) and various deletion mutants (#2-4). F , Immunoprecipitation and western blot assays using Flag antibody showing the interaction between p65 and LTF protein in U251 cells transfected with a series of truncations of Flag-tagged LTF. G , Schematic representation of Flag-tagged full-length p65 (#1) and various deletion mutants (#2-5). H , Immunoprecipitation and western blot assays using Flag antibody showing the interaction between p65 and LTF protein in U251 cells transfected with a series truncations of Flag-tagged p65.

Article Snippet: Antibodies against LTF (10933-1-AP), Cyclin E1 (11554-1-AP), Cyclin D1 (26939-1-AP), E-cadherin (20874-1-AP), N-cadherin (22018-1-AP), γ-H2AX (29380-1-AP), PP2A (13482-1-AP), GAPDH (10494-1-AP), and H3 (17168-1-AP) were from Proteintech (China).

Techniques: Phospho-proteomics, Control, Western Blot, Transfection, Expressing, Immunoprecipitation

Journal: bioRxiv

Article Title: A live tumor fragment platform to assess immunotherapy response in core needle biopsies while addressing challenges of tumor heterogeneity

doi: 10.1101/2025.07.18.663728

Figure Lengend Snippet: A Schematic of protocol used to validate sequential treatment strategy. Tumor resections were used due to the availability of sufficient tissue to address inter-well heterogeneity in tissue composition and enable meaningful cross-well comparisons. Tumor resections were cut into resection LTFs, encapsulated in hydrogel and treated with IgG for 28 hours (shown in blue), positive control αCD3/αCD28 to stimulate T cells for 28 hours (shown in red), or IgG4 for the first 20 hours and αCD3/αCD28 for the final 8 hours of culture (shown in red). Conditioned media was sampled from each of the wells at 4, 20, and 28 hours for cytokine profiling. B IFNγ induction in a head and neck human tumors specimen. IFNγ and CXCL10 induction was observed following αCD3/αCD28 stimulation in cross-well (left) and sequential treatment (right). IL-1Ra remained unchanged following αCD3/αCD28 stimulation. C To characterize the similarity in cytokine production following the cross-well and sequential treatment experimental designs, the fold change in rates (slopes) of cytokine production following αCD3/αCD28 stimulation were calculated compared to rates of cytokine production during IgG exposure. The fold changes (FC) in rates of cytokine production for cross-well and sequential treatment strategies were compared to determine similarities of magnitude and direction (Increase >1.5; no change = 0.5-1.5; decrease <0.5). IFNγ, CXCL10 and IL-1Ra showed 100%, 83% and 83% concordance, respectively, between cross-well and sequential treatment strategies. Numerical labels are shown in cases when cross-well and sequential treatment data are discordant. Grey band indicates no change.

Article Snippet: Humanized non-small cell lung cancer patient-derived xenograft (PDX) tumors were generated by warm-passaging 300 μm x 300 μm x 300 μm PDX LTFs into the hind flank of female NSG mice (The Jackson Laboratory) between 7-9 weeks-old.

Techniques: Positive Control

Journal: bioRxiv

Article Title: A live tumor fragment platform to assess immunotherapy response in core needle biopsies while addressing challenges of tumor heterogeneity

doi: 10.1101/2025.07.18.663728

Figure Lengend Snippet: A Schematic shows the workflow for creating LTFs from human tumor resections used for assessing the ex vivo response to treatment. Tumor resections were fragmented using a specialized cutting instrument to create LTFs, encapsulated in a hydrogel and treated with either an ICI (eg, αPD-1), a positive control for T-cell stimulation (eg, αCD3/αCD28), or IgG control, followed by cytokine profiling to assess response to treatment. B IHC image of a CD3 stained human melanoma resection showing a standard slice sampling technique (left) compared to LTF 300 µm x 300 µm segments (right). C In silico randomized pools of large slices from the melanoma resection showing significantly greater variability in CD3+ staining when compared to randomized pools of LTFs cut to 300 µm edge lengths. D Effect of LTF number on the percent of cytokines in the assay panel with measured concentrations exceeding the lower limit of quantitation (LLOQ) following stimulation with αCD3/αCD28 for 24 hours. Data are reported for 5 human tumor specimens (ovarian, n=1; lung, n=1; colorectal, n=1; head and neck, n=2). E Variability (CV (%)) across 5 replicate wells of 200 resection LTFs (300 µm (length) x 300 µm (width) x 300 µm (depth)) from each of 10 human tumors (pancreatic, n=2; head and neck, n=4; ovarian, n=3; triple negative breast, n=1) stimulated with αCD3/αCD28. Cytokines of particular interest are CXCL10 and IFNγ (highlighted in red) due to their increase in secretion with T-cell stimulation. * P < 0.05, ** P < 0.005

Article Snippet: Humanized non-small cell lung cancer patient-derived xenograft (PDX) tumors were generated by warm-passaging 300 μm x 300 μm x 300 μm PDX LTFs into the hind flank of female NSG mice (The Jackson Laboratory) between 7-9 weeks-old.

Techniques: Ex Vivo, Positive Control, Cell Stimulation, Control, Staining, Sampling, In Silico, Quantitation Assay

Journal: bioRxiv

Article Title: A live tumor fragment platform to assess immunotherapy response in core needle biopsies while addressing challenges of tumor heterogeneity

doi: 10.1101/2025.07.18.663728

Figure Lengend Snippet: B H&E-stained sections of a colorectal cancer resection showing low grade adenocarcinoma. Lymphocytes (red arrows), fibroblasts (blue arrow), tumor cells (green arrow) and collagen fibers (yellow arrow) are present in both the intact resection specimen (A) and the resection LTFs at 4 hours (B). Comparison of H&E images by a board-certified pathologist identified conservation of histology including intratumor heterogeneity. C H&E-stained sections of head and neck LTFs at 4 hours and 48 hours of ex vivo culture showing tumor cells (green arrow), collagen fibers (yellow arrow) and lymphocytes (red arrow). D-F Comparison of the percentages of CD4+ ( D ), CD8+ ( E ), and CD4+ to CD8+ cell ratios ( F ) at 4 hours vs. 48 hours as assessed by IHC (N=9 human tumors; colorectal, n=1; bladder, n=1; head and neck, n=2; ovarian, n=3; pancreatic, n=1; uterine, n=1). G Changes in viability (CCK8 assay) over 72 hours in culture of LTFs derived from 5 human tumor resections. H Changes in T cell function over 72 hours of culture of LTFs as assessed by IFNγ production following 24 hours of stimulation with αCD3/αCD28. * P < 0.05.

Article Snippet: Humanized non-small cell lung cancer patient-derived xenograft (PDX) tumors were generated by warm-passaging 300 μm x 300 μm x 300 μm PDX LTFs into the hind flank of female NSG mice (The Jackson Laboratory) between 7-9 weeks-old.

Techniques: Staining, Comparison, Ex Vivo, CCK-8 Assay, Derivative Assay, Cell Function Assay

Journal: bioRxiv

Article Title: A live tumor fragment platform to assess immunotherapy response in core needle biopsies while addressing challenges of tumor heterogeneity

doi: 10.1101/2025.07.18.663728

Figure Lengend Snippet: Cross-well comparison of cytokine production in response to ICI treatment is not amenable to CNBs due to limited tissue availability and inability to address intra-specimen heterogeneity . A Schematic showing the distribution of tissue from a core needle biopsy (CNB) in an experiment using the cross-well configuration where tissue amount is insufficient to address intra-specimen heterogeneity. B The percentage of cytokines out of a 30-cytokine panel with concentrations above LLOQ for the cross-well configuration for resection LTFs (Resection cross-well) and CNB LTFs (CNB cross-well) following treatment with IgG control for 24 hours. There is no significant difference in the ability to detect cytokines between the two form factor protocols (ns = not significant, P = 0.089). C The variability in concentrations of cytokines from resection LTFs and CNB LTFs plated in the cross-well configuration and treated with ICI for 24 hours. There is significantly greater variability in cytokine concentrations between replicate wells of treated groups in CNB LTFs compared to resection LTFs. **** P < 0.0001

Article Snippet: Humanized non-small cell lung cancer patient-derived xenograft (PDX) tumors were generated by warm-passaging 300 μm x 300 μm x 300 μm PDX LTFs into the hind flank of female NSG mice (The Jackson Laboratory) between 7-9 weeks-old.

Techniques: Comparison, Control

Journal: bioRxiv

Article Title: A live tumor fragment platform to assess immunotherapy response in core needle biopsies while addressing challenges of tumor heterogeneity

doi: 10.1101/2025.07.18.663728

Figure Lengend Snippet: A Schematic shows the workflow for creating LTFs from human CNBs used for assessing the ex vivo response to treatment. Tumor CNBs are sliced at a 20° angle using a specialized cutting instrument to create LTFs which are then encapsulated in a proprietary hydrogel (referred to as Elephas hydrogel hereafter) for ex vivo culture, enabling cytokine profiling, histological analysis and advanced imaging. B The spatial stability of CNB LTFs created from CT26 syngeneic tumors (N=4 tumors) over 48 hours of ex vivo culture when encapsulated in VitroGel ® -3 versus Elephas hydrogel. Circles highlight regions of hydrogel where LTF stability is compromised with VitroGel-3 encapsulation but not with Elephas hydrogel. C The distances between CNB LTF locations at 72 hours vs. 4 hours of culture were measured and reported relative to 4 hours when encapsulated in Elephas hydrogel or VitroGel ® -3. Limited movement was observed with Elephas hydrogel, highlighting improved stability and enabling longitudinal advanced imaging. D-E Dynamic optical coherence microscopy (dOCM) imaging of a human lung (NSCLC) CNB LTF at 4 hours ( D ) and 48 hours ( E ) in culture. Volume positive for metabolic activity indicating tissue viability is false-colored in green and volume negative for metabolic activity is false-colored in magenta. Note that volume negative for metabolic activity represents non-viable and acellular tissue. F The ratios of viable voxels to total voxels are reported across 3 human tumors (lung, breast, kidney) and show maintenance of viability over 48 hours in culture. G The ratio of viable volume (measured in voxels) to total volume reported for 6 CNB LTFs from Lung 5793 highlights heterogeneity in viability across LTFs from a single CNB. H A significant decrease in the number of CD3+ cells, normalized to area, is seen in unencapsulated compared to encapsulated humanized PDX CNB LTFs after 48 hours in culture (each symbol represents 1 tumor, N=5 tumors per group, * P < 0.05). I Representative CD3-labeled sections of humanized PDX CNB LTFs from encapsulated (top) and unencapsulated (bottom) tissues after 48 hours of ex vivo culture. J-K Multiplex immunofluorescence labeled fragments from Liver 5605 show the presence of tumor cells (PanCK), macrophages (CD68), T cells (CD3), stroma/fibroblasts (SMActin) and cell nuclei (DAPI) at 4 hours ( J ) and 48 hours ( K ) in culture.

Article Snippet: Humanized non-small cell lung cancer patient-derived xenograft (PDX) tumors were generated by warm-passaging 300 μm x 300 μm x 300 μm PDX LTFs into the hind flank of female NSG mice (The Jackson Laboratory) between 7-9 weeks-old.

Techniques: Ex Vivo, Imaging, Encapsulation, Microscopy, Activity Assay, Labeling, Multiplex Assay, Immunofluorescence

Journal: bioRxiv

Article Title: A live tumor fragment platform to assess immunotherapy response in core needle biopsies while addressing challenges of tumor heterogeneity

doi: 10.1101/2025.07.18.663728

Figure Lengend Snippet: A Encapsulated LTFs from CNBs are sequentially treated with IgG control followed by ICI. Conditioned media is collected at the ∼4-, ∼20-and ∼48-hour time points to enable measurement of cytokine concentrations which are used to calculate the rate of cumulative cytokine production during the control (4-20 hours) and ICI (20-48 hours) treatment phases. Fold change in the rate of cytokine production (slope of ICI treatment phase / slope of IgG control phase) can be used to assess ex vivo treatment response. Exemplary human lung ( B , Lung 4624 treated with αPD-1), colon ( E , Colon 4834 treated with αPD-L1) and kidney ( H , Kidney 5023 treated with αPD-1 + αCTLA-4) cancer CNBs exhibiting an increase in rate of IFNγ and CXCL10 production following ICI treatment suggesting a potential cytokine response. Exemplary human lung ( C , LU4578 treated with αPD-1), esophageal ( F , Esophageal 4798 treated with αPD-L1) and kidney ( H , Kidney 5327 treated with αPD-1 + αCTLA-4) cancer CNBs exhibiting a similar rate of IFNγ and CXCL10 production between the ICI and IgG treatment phases suggesting an unlikely cytokine response. Note that multiple CNBs were obtained from patients 4624, 4578 and 4834 enabling profiling of replicate wells. The fold change in the slope of the ICI treatment phase compared to the slope in the IgG treatment phase for IFNγ and CXCL10 across 7 lung ( D ), 2 gastrointestinal ( G ) and 6 kidney ( J ) cancer CNBs. Individual data points report maximum fold change for those specimens where multiple CNBs/replicate wells were profiled. Note that the maximum fold change for the exemplary cytokine responsive specimens (Lung 4624, Colon 4834 and Kidney 5023, shown in red) is at the top of the distribution while the maximum fold change for the exemplary cytokine nonresponsive specimen (Lung 4578, Esophageal 4798 and Kidney 5327, shown in blue) is close to 1.

Article Snippet: Humanized non-small cell lung cancer patient-derived xenograft (PDX) tumors were generated by warm-passaging 300 μm x 300 μm x 300 μm PDX LTFs into the hind flank of female NSG mice (The Jackson Laboratory) between 7-9 weeks-old.

Techniques: Control, Ex Vivo