Molecular drivers of neutrophil recruitment to primary non-small cell lung cancer
Bibliographic record
Abstract
Neutrophils are associated with developing cancer lesions and are the main immune component of primary non-small cell lung cancer (NSCLC). Multiple studies support the notion that tumor associated neutrophils (TANs) can promote tumor progression. However, the exact mechanism in which these neutrophils are recruited to the tumor microenvironment remains unclear. We hypothesize that there is a hierarchy of molecular cues produced by developing lung cancers that guide circulating neutrophils to infiltrate the tumor microenvironment. Identifying these cues may permit modulation of neutrophil infiltration within developing lung cancers and act as an immunotherapeutic tool to suppress cancer progression and improve response to existing therapeutics. To this end, we profiled H-59-GFP Lewis lung carcinoma injected mice and control mice using a qRT-PCR 84 gene panel. We also profiled 3 established human primary NSCLC cell lines representing common NSCLC subtypes in patients (A549; KRAS mutant, PC9; EGFR mutant, HCC78; ROS1 translocation). We focused on 2 of the most upregulated genes across our cell lines, macrophage inhibitory factor (MIF) and C-X-C motif ligand 1 (CXCL1). Most notably, CXCL1 was upregulated 13-fold in our H-59 injected mouse versus control. After confirming protein expression of these targets by western blot and ELISA, we performed shRNA knock down (KD) of these genes and tested the migration of neutrophils towards treated and control cell lines using a novel microfluidic device. Findings from KD experiments were confirmed via antibody-mediated inhibition in the chance of non-specific KDs. We observed a 3-fold increase of neutrophil migration towards A549 cancer cell line, scramble, and empty vector controls compared to our serum free (SF) negative control (p<0.0001). This increase was inhibited in our MIF (56% decrease, p=0.0002) and CXCL1 (72% decrease, p=0.0018) KDs. Repetition with antibody-mediated inhibition yielded similar results; neutrophil migration was inhibited utilizing MIF neutralizing antibody (N.A.) (58% decrease, p=0.0122) and CXCL1 N.A. (59% decrease, p<0.0001) relative to our A549 cancer cell line. We have therefore identified 2 proteins that play a key role in neutrophil recruitment to NSCLC cell lines in vitro and have demonstrated the application of a novel microfluidic device to analyze neutrophil recruitment. Our data provides the basis for in vivo investigations to elucidate the key molecular cues for neutrophil infiltration within developing lung cancers.
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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 | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.000 |
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".