Neutrophil-mediated fibroblast-tumor cell il-6/stat-3 signaling underlies the association between neutrophil-to-lymphocyte ratio dynamics and chemotherapy response in localized pancreatic cancer: A hybrid clinical-preclinical study
- Department of Surgery, University of Miami Miller School of Medicine, United States
- Department of Surgery, University of Nebraska Medical Center, United States
- Sylvester Comprehensive Cancer Center, United States
- Department of Pathology, University of Miami, United States
- Department of Medicine, University of Miami, United States
- Department of Public Health Sciences, The University of Texas Health Science Center at Houston, United States
Figures
Figure 1
Neutrophil-to-lymphocyte ratio (NLR) dynamics are associated with pathologic response and survival following neoadjuvant chemotherapy in pancreatic cancer.
(A) STROBE diagram for selection of study-eligible patients with potentially operable pancreatic ductal adenocarcinoma undergoing neoadjuvant chemotherapy and curative-intent pancreatectomy, …
Figure 2
Attenuating neutrophil-to-lymphocyte ratio (NLR) improves sensitivity to chemotherapy in preclinical models of pancreatic cancer.
(A) Schematic of in vivo experimental design, illustrating treatment groups utilized (vehicle, anti-Ly6G [αLy6G] alone, gemcitabine/paclitaxel alone, and gemcitabine/paclitaxel+αLy6G), treatment …
Figure 3
Improved chemosensitivity following neutrophil attenuation in pancreatic cancer is associated with anti-tumor adaptive immunity.
(A) viSNE maps depicting comparison of splenocyte-derived circulating Ly6G+ (left) and Ly6C+ (right) MDSCs gated within Cd11b+F4/80 cells in NLR-attenuating anti-Ly6G (25 μg) vs. vehicle-treated KPC …
Figure 4
Improved chemosensitivity with attenuated NLR is associated with reduction in inflammatory CAF abundance and IL-6/STAT-3 signaling in the tumor microenvironment.
(A) Representative contour plots of CD45-CD31-PDPN+ cancer-associated fibroblasts (CAF) gated on Ly6C and MHC-II across vehicle, NLR-attenuating αLy6G, gemcitabine plus paclitaxel (Gem/Pac) alone, …
Figure 5
Neutrophil-derived IL-1β induces pancreatic fibroblast-tumor cell IL-6/STAT-3 signaling.
(A) Bubble plot representing multiplex cytokine array performed on condition media from column-sorted Ly6G+F4/80- neutrophils (24-hr culture) derived from whole pancreata of KPC orthotopic mice. The …
Appendix 1—figure 1
NLR dynamics during neoadjuvant chemotherapy (NAC) are associated with pathologic response in patients undergoing resection for pancreatic cancer.
Area under the receiver operating characteristic curve (AUC) statistics estimating the predictive capacity of three biomarker models (pre-chemotherapy aNLR only, ΔNLR only, combined model aNLR + …
Appendix 1—figure 2
NLR dynamics during neoadjuvant chemotherapy (NAC) are associated with survival in patients undergoing resection for pancreatic cancer.
Time-dependent AUC analysis with internal bootstrap validation examining the three biomarker models for (A) disease-free survival (DFS; left) and (B) overall survival (OS; left) for years 1–3. …
Appendix 1—figure 3
Titration of non-depleting NLR attenuating anti-Ly6G antibody dosing in a preclinical model of PDAC.
(A) Representative flow cytometry contour plots depicting circulating splenocyte-derived Ly6G+ (top) and CD3+ (bottom) cell populations 2 weeks following anti-Ly6G dose titration experiment to …
Appendix 1—figure 4
NLR attenuation with or without systemic chemotherapy in preclinical models of PDAC does not increase treatment-related toxicity.
(A) Mean (± standard deviation) body weights, and (B) mean ± SD of alanine transferase (ALT) levels from blood of mice in each cohort (n=8–10/group) graphed at 3 time points based on treatment …
Appendix 1—figure 5
PDPN is a marker of pancreatic tumor-associated cancer associated fibroblasts (CAF).
(A) Gating strategy for flow cytometric analysis of PDPN+ CAF populations after exclusion of CD45+, EpCAM+, and CD31+ cells; (B) Dot plot from single cell RNA sequencing (scRNAseq) dataset …
Appendix 1—figure 6
Neutrophil-derived IL-1β is a novel mediator of inflammatory CAF polarization in pancreatic cancer.
Schematic of experimental design illustrating ex vivo co-culture of KPC CAFs with intratumoral column-sorted Ly6G+F4/80 cells from whole pancreata of KPC orthotopic mice, with or without …
Appendix 1—figure 7
Anti-Ly6G antibody specifically targets PDAC-associated neutrophils, but not other granulocytic populations.
(A) Schematic of experimental design showing timing/dosing of anti-Ly6G treatment in orthotopic KPC tumor-bearing mice (top), with adjacent histograms showing (% of parent CD45+ cells) relative …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Mus musculus) | Pancreatic Tumor Cells from KrasLSL-12D/+;Trp53R172H/+;Pdx1Cre (KPC) mouse | Ben Stanger/UPenn | KPC6694c2 | |
| Cell line (Mus musculus) | Tumor associated fibroblasts from KPC mouse | Nagathihalli et al., 2016 | KPC CAFs | |
| Other | Ptf1aCre/+;KrasLSL-G12D/+;Tgfbr2flox/flox | Datta et al., 2022 | PKT | Genetically engineered mouse |
| Antibody | Anti-Ly6G (Rat monoclonal) reactive to mouse | BioXcell | Clone 1A8 Catalog# BE0075-1 | 25 μg/dose |
| Antibody | Anti-IL-1β neutralizing antibody (E. coli, polyclonal) | R&D Systems | Catalog# AF-401-NA | 1:80 |
| Antibody | Cxcl1 (Rabbit, monoclonal) Reactive to human and mouse | Abcam | Catalog# ab86436 | 1:500 |
| Antibody | Podoplanin (Mouse, monoclonal) Reactive to human | Cell Signalling | Catalog# 26981 | 1:200 |
| Antibody | Podoplanin (Syrian hamster, monoclonal) Reactive to mouse | Abcam | Catalog# ab92319 | 1:200 |
| Antibody | CD3 (170Er, Human, monoclonal) 3170019D | Fluidigm | 3170019D | 1:1000 |
| Antibody | CD11B (149Sm, Human, monoclonal) | Fluidigm | 3149028D | 1:1000 |
| Antibody | α-SMA (141Pr, Human, monoclonal) | Fluidigm | 314017D | 1:1000 |
| Antibody | Pan-Cytokeratin (148Nd, Human, monoclonal) | Fluidigm | 3148022D | 1:1000 |
| Antibody | CD15 (164Dy, Human, monoclonal) | Fluidigm | 3164001B | 1:1000 |
| Antibody | CD8 (146Nd, Human, monoclonal) | Fluidigm | 3146001B | 1:1000 |
| Chemical compound, drug | Anakinra | SOBI Pharmaceuticals | α-IL-1R1 inhibitor | |
| Sequence-based reagent | Cxcl1 Primer - Mouse | Qiagen | Gene ID - QT00115647 | |
| Sequence-based reagent | Il6 Primer - Mouse | Qiagen | Gene ID - QT00098875 | |
| Commercial assay or kit | Cytokine array - Mouse | R&D Systems | ARY006 |
Appendix 1—table 1
Clinicopathological characteristics of study-eligible patients with localized pancreatic ductal adenocarcinoma who received neoadjuvant chemotherapy (NAC) and underwent curative-intent pancreatectomy (BMI: body mass index; HRD: Homologous Recombination Deficiency; ECOG: Easter Cooperative Oncology Group; CAP: College of American Pathology; G-CSF: Granulocyte-colony stimulating factor).
| Variable | All patients (n=94) | Complete/ partial response to NAC (n=66) | Absent/poor response to NAC(n=28) | P-value |
|---|---|---|---|---|
| Age (mean ± SD) | 67.3±10.3 | 67.5±10.9 | 66.9±8.9 | 0.78 |
| Female gender, n (%) | 58 (61.7 %) | 41 (62.1%) | 17 (60.7%) | 0.89 |
| Diagnosis BMI, (mean ± SD) | 26.7±4.9 | 26.8±5 | 26.4±4.6 | 0.68 |
| Hispanic Ethnicity, n (%) | 43 (45.8%) | 30 (45.5%) | 13 (46.4%) | 0.64 |
| Germline HRD mutation, n (%) Germline HRD mutation No germline HRD mutation | 6 (6.3%) 88 (93.7%) | 6 (9.1%) 60 (91.9%) | 0 (0%) 28 (100%) | 0.17 |
| Pre-chemotherapy Absolute Blood Counts Total Leukocyte count (103 /μl) Neutrophil count (%) Lymphocyte count (%) Platelet count (103 /μl) | 7.2±1.6 65.3±7.3 22.3±7.0 243±71.5 | 7.2±1.8 63.7±6.9 24.8±6.7 246±69.5 | 7.25±1.5 69.7±4.1 17.6±3.5 234±77.3 | 0.92 <0.001 <0.001 0.59 |
| Pre-chemotherapy Neutrophil/Lymphocyte Ratio (median ± SD) | 3.0±1.3 | 2.5±1.0 | 3.9±1.2 | <0.001 |
| Diagnosis CA 19–9 levels (median ± SD) | 181±1,823 | 147±923 | 202±2,944 | 0.50 |
| Pre-Surgery Absolute Blood Counts Total Leukocyte count (103 /ul) Neutrophil count (%) Lymphocyte count (%) Platelet count (* 103 /ul) | 7±1.92 63.2±9.5 23.6±7.7 198±74.1 | 6.1±1.72 60±7.8 26.5±6.4 203±57.8 | 8.5±1.95 71±6.25 16.7±4.7 181±104 | 0.01 <0.001 <0.001 0.03 |
| Pre-Surgery Neutrophil/ Lymphocyte Ratio (median ± SD) | 2.6±1.57 | 2.3±0.78 | 4.2±1.68 | <0.001 |
| ΔNLR (=Pre-Surgery-Pre-Chemo NLR) (median ± SD) | - 0.1±1.25 | - 0.38±1.1 | 0.21±1.5 | 0.01 |
| ECOG Status, n (%) 0 1 2 | 36 (38.3%) 49 (52.1%) 9 (9.6%) | 27 (40.9%) 34 (51.5%) 5 (7.6%) | 9 (32.1%) 15 (53.6%) 4 (14.3%) | 0.51 |
| Tumor location Head Body Tail | 76 (80.8%) 9 (9.6%) 9 (9.6%) | 53 (80.3%) 6 (9.1%) 7 (10.6%) | 23 (82.1%) 3 (10.7%) 2 (7.2%) | 0.96 |
| Resectability status Resectable Borderline resectable Locally advanced | 21 (22.3%) 50 (53.2%) 23 (24.5%) | 15 (22.7%) 36 (54.6%) 15 (22.7%) | 6 (21.4%) 14 (50.0%) 8 (28.6%) | 0.83 |
| Radiographic tumor size (median ± SD) | 30±14.2 | 30±10.7 | 28.5±20.2 | 0.19 |
| Neoadjuvant Chemotherapy Gemcitabine/Abraxane FOLFIRINOX Both | 35 (37.2%) 49 (52.2%) 10 (10.6%) | 26 (39.4%) 33 (50.0%) 7 (10.6%) | 9 (32.1%) 16 (57.1%) 3 (10.8%) | 0.79 |
| Duration of NAC (months)* | 4±2.3 | 4±2.2 | 4±2.6 | 0.22 |
| Use of G-CSF during NAC | 91 (96.8%) | 64 (97.0%) | 27 (96.4%) | 1.00 |
| Neoadjuvant radiation | 6 (6.4%) | 4 (6.1%) | 2 (7.1%) | 1.00 |
| Histology grade† Well Differentiated Moderately differentiated Poorly differentiated | 2 (2.1%) 59 (62.8%) 27 (28.7%) | 2 (3%) 49 (74.2%) 11 (16.7%) | 0 (0%) 10 (35.7%) 16 (57.1%) | 0.002 |
| pT classification T1 T2 T3 T4 | 27 (28.7%) 33 (35.1%) 28 (29.8%) 6 (6.4%) | 27 (40.9%) 22 (33.3%) 14 (21.3%) 3 (4.5%) | 0 (0%) 11 (39.3%) 14 (50.0%) 3 (10.7%) | <0.001 |
| pN classification Positive Negative | 52 (55.3%) 42 (44.7%) | 32 (48.5%) 34 (51.5%) | 20 (71.4%) 8 (28.6%) | 0.04 |
| Pathological Stage IA IB IIA IIB III IV | 17 (18.1%) 12 (12.8%) 10 (10.6%) 47 (50%) 7 (7.4%) 1 (1.1%) | 17 (25.7%) 9 (13.6%) 6 (9.1%) 30 (45.5%) 4 (6.1%) 0 (0%) | 0 (0%) 3 (10.7%) 4 (14.3%) 17 (60.7%) 3 (10.7%) 1 (3.6%) | 0.04 |
| Neoadjuvant therapy response (CAP grading)‡ Grade 0 Grade 1 Grade 2 Grade 3 | 0 (0%) 12 (12.8%) 54 (57.4%) 28 (29.8%) | - 12 (18.2%) 54 (81.8%) - | 28 (100%) | N/A |
| R0 resection margin Yes No (R1 resection) | 78 (83%) 16 (17%) | 61 (92.4%) 5 (7.6%) | 17 (60.7%) 11 (39.3%) | <0.001 |
| Adjuvant therapy, n (%) | 58 (61.7%) | 43 (65.2%) | 15 (53.6%) | 0.29 |
| Local Recurrence Yes No | 19 (20.2%) 75 (79.8 %) | 10 (15.2%) 56 (84.8%) | 9 (32.1%) 19 (67.8%) | 0.06 |
| Distant Recurrence Yes No | 55 (58.5%) 39 (41.5%) | 34 (51.5%) 32 (48.5%) | 21 (75%) 7 (25%) | 0.03 |
-
*
Due to variation in dose scheduling between FOLFIRINOX and gemcitabine/abraxane, duration of NAC is reported in months (vs. number of cycles)
-
†
Grade information missing in 6 patients
-
‡
College of American Pathologist (CAP) grading: Grade 0, no viable residual tumor (pathologic complete response); Grade 1, marked response (minimal residual cancer with single cells or small groups of cancer cells); Grade 2, partial response (residual cancer with evident tumor regression, but more than single cells or rare small groups of cancer cells); and Grade 3, poor or no response (extensive residual cancer with no evident tumor regression)
Appendix 1—table 2
Salient clinical characteristics and single-cell image segmentation details from imaging mass cytometry experiments comparing tissue-level neutrophil-to-lymphocyte ratio (NLR) and stromal α-SMA pixel intensity in pre-chemotherapy tissue sections from localized pancreatic ductal adenocarcinoma (PDAC) patients who demonstrated either partial/complete or poor/absent pathologic response to neoadjuvant chemotherapy.
| Pt # | Primary tumor | NAC regimen | Duration of NAC (mo) | Neoadjuvant radiation | Pathologic response | # total single cells in IMC slide | #CD11b+CD15+ neutrophils | #CD3+CD8+ T cells | NLR (norm. to 5000 cells) |
|---|---|---|---|---|---|---|---|---|---|
| 1 | Borderline Resectable | FFX | 4 | No | Partial (CAP 2) | 4326 | 325 | 29 | 11.2 |
| 2 | Borderline Resectable | FFX | 4.5 | No | Partial (CAP 2) | 3256 | 256 | 32 | 7.9 |
| 3 | Locally Advanced* | GNP | 6 | No | Near-complete (CAP 1) | 9421 | 472 | 142 | 3.3 |
| 4 | Borderline Resectable | GNP | 5 | No | Poor/Absent (CAP 3) | 8621 | 951 | 61 | 15.6 |
| 5 | Locally Advanced* | FFX | 6 | No | Poor/Absent (CAP 3) | 6011 | 1105 | 59 | 18.7 |
| 6 | Resectable | FFX +GNP | 6 | No | Poor/Absent (CAP 3) | 4712 | 523 | 40 | 13.1 |
-
*
Representative tissue and image segmentation maps depicted in Figure 4
Appendix 1—table 3
Predictors of partial/complete pathologic response following neoadjuvant chemotherapy in resected patients with localized pancreatic ductal adenocarcinoma using multivariable logistic regression.
| Variable | OR (95% CI) | P-value |
|---|---|---|
| Age | 1.02 (0.92, 1.09) | 0.68 |
| Gender Female Male | Ref 2.86 (0.45, 26.2) | - 0.34 |
| Diagnosis BMI | 0.88 (0.74, 1.08) | 0.21 |
| CA 19–9 dynamics Any increase Any decrease | Ref 1.82 (0.001, 3.74) | - 0.05 |
| Resectability Status Borderline Locally advanced Resectable | Ref 2.72 (0.22, 33.2) 0.64 (0.06, 7.44) | - 0.43 0.72 |
| Radiographic tumor size | 0.98 (0.91, 1.05) | 0.54 |
| NAC duration (months) | 1.09 (0.68, 1.75) | 0.73 |
| Absolute pre-chemotherapy aNLR Low High | Ref 0.02 (0.003, 0.15) | <0.001 |
| ΔNLR Low High | Ref 0.06 (0.01, 0.33) | 0.002 |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/78921/elife-78921-mdarchecklist1-v2.docx
-
Source data 1
Clinical Cohort and Western Blotting Source Data.
- https://cdn.elifesciences.org/articles/78921/elife-78921-data1-v2.zip
