Additional file 1 of TNFα secreted by glioma associated macrophages promotes endothelial activation and resistance against anti-angiogenic therapy
Bibliographic record
Abstract
Additional file 1. Figure S1. RT-PCR angiogenesis array Human umbilical vein cells (HUVEC) were incubated with media alone (EBM) or conditioned medium (CM) from RAW264.7 macrophages stimulated with normal human astrocytes (Mφ-NHA) or GBM cell lines U87, U118, U251, A172 (Mφ-U87, Mφ-U118, Mφ-U251, Mφ-A172). mRNA was extracted and analyzed by an RT-PCR angiogenesis array. Results were normalized to RPL30 and ACTB. The relative quantity of significantly altered genes (> 2-fold relative to Mφ-NHA or Mφ-NSC) are shown. n = 3, mean ± max/min, 95% confidence interval. Figure S2. Non-Glioma associated macrophages do not activate ECs Medium alone (EBM) or CM from normal human astrocytes (Mφ-NHA), GBM cell line (Mφ-U87), renal cell adenocarcinoma cell line (Mφ-786-O), human non-small cell carcinoma cell line (Mφ-H1299), transformed Schwanoma cell line (Mφ-Hei193) or osteosarcoma cell line (Mφ-U2OS) were analyzed by a multi-analyte inflammatory ELISA array. Only the U87 GBM cell line induces upregulation of VCAM1, ICAM1, CXCL5 and CXCL10. n = 3, mean ± max/min, 95% confidence interval. *p < 0.05. Figure S3. RT-PCR angiogenesis array Human cerebral ECs (hCMEC/D3) were incubated with media alone (EBM) or conditioned medium (CM) from RAW264.7 macrophages stimulated with normal human astrocytes (Mφ-NHA) or GBM cell lines U87 (Mφ-U87). mRNA was extracted and analyzed by an RT-PCR angiogenesis array. Results were normalized to RPL30 and ACTB. The relative quantity of significantly altered genes are shown. n = 3, mean ± max/min, 95% confidence interval. Figure S4. VEGF RT-PCR assay Human umbilical vein cells (HUVEC) were incubated with media alone (EBM) or conditioned medium (CM) from RAW264.7 macrophages stimulated with normal mouse astrocytes (Mφ-NMA) or GL-261 cell lines (Mφ-GL261). mRNA was extracted and analyzed by an RT-PCR with VEGF gene. Results were normalized to RPL30 and ACTB. The relative quantity of VEGF is shown (n=3, mean ± max/min, 95% confidence interval). Figure S5. Expression of alpha 4 integrin in tumor microenvironment and on cells a) IHC with alpha 4 integrin has been performed on tumor sections from GL261 syngeneic mouse model treated with vehicle control or TNFα inhibitor, MP6-XT22. b) Flow cytometry analysis todetermine the expression of alpha 4 integrin on Raw264.7 cells and U973 cells as a positive control. Anti-Integrin alpha 4/CD49D antibody (ab202969) was diluted in 1:100 for both IHC and Flow cytometry. Alexa 488 donkey anti-rabbit secondary antibody (A32790) was diluted in 1:1000.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.002 | 0.030 |
| Meta-epidemiology (narrow) | 0.001 | 0.001 |
| Meta-epidemiology (broad) | 0.002 | 0.001 |
| Bibliometrics | 0.002 | 0.004 |
| Science and technology studies | 0.001 | 0.000 |
| Scholarly communication | 0.003 | 0.002 |
| Open science | 0.002 | 0.001 |
| Research integrity | 0.002 | 0.001 |
| Insufficient payload (model declined to judge) | 0.902 | 0.176 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".