Lung cancer-fueled emergency myelopoiesis is characterized by an increase of S100A9+ and LCN2+ hematopoietic stem and progenitor cells

This study reveals that lung cancer drives emergency myelopoiesis in the bone marrow by expanding S100A9+ and LCN2+ hematopoietic stem and progenitor cells, where S100A9 acts as an upstream regulator of LCN2 and represents a promising therapeutic target to inhibit tumor growth and overcome immunotherapy resistance.

The Gist

Imagine your body as a bustling city. The Bone Marrow is the city's central factory where all the security guards (immune cells) are built. Usually, this factory runs on a steady schedule, producing just enough guards to keep the peace.

Now, imagine a Lung Cancer tumor is like a rogue gang setting up shop in the city's lungs. This gang doesn't just stay put; they send out urgent, chaotic radio signals to the central factory.

Here is what this paper discovered about what happens next, explained simply:

1. The Factory Goes into "Emergency Mode"

When the lung cancer gang sends its distress signals, the Bone Marrow factory goes into panic mode. Instead of building a balanced mix of guards, it starts churning out raw, untrained recruits (immature stem cells) at a frantic pace.

• The Analogy: Think of a bakery that usually makes perfect, ready-to-eat loaves of bread (mature immune cells). When the alarm rings, the bakery stops baking the finished loaves and just starts dumping bags of raw dough (immature cells) onto the counter. They have too much raw material, but not enough finished products to actually fight the gang.
• The Result: The factory gets clogged with these raw recruits, while the city's lungs get flooded with immature, confused guards who can't fight effectively. In fact, some of these immature guards actually help the cancer gang by suppressing the body's real defenses.

2. The "Alarm Bells" (S100A9 and LCN2)

The researchers found two specific proteins acting like loud, persistent alarm bells throughout this entire process.

• S100A9 is like the Master Alarm. It's the signal that tells the factory to keep producing raw dough.
• LCN2 is like the Second Alarm that rings right after the first one. The study found that S100A9 actually causes LCN2 to ring. They work as a team to keep the factory in chaos.

These alarms are so loud that they are heard not just in the factory (bone marrow), but also in the blood and the tumor itself.

3. The "Traffic Jam" in the Factory

Usually, once the guards are built, they leave the factory to patrol the city. But in this cancer scenario, the factory's exit doors are jammed.

• The cancer signals mess with the "doorman" (a protein called CXCR4). The doorman forgets to open the gates, so the raw recruits get stuck inside the factory.
• This explains why the factory looks so crowded with raw materials, even though the body is desperate for finished guards.

4. The Solution: Turning Off the Master Alarm

The researchers tested a drug called Tasquinimod. Think of this drug as a mute button for the Master Alarm (S100A9).

• What happened? When they hit the mute button:
  1. The factory stopped panicking and started making finished guards again.
  2. The cancer tumor in the lungs started to shrink.
  3. It worked even when combined with standard immunotherapy (which tries to wake up the immune system).

Interestingly, the standard immunotherapy (anti-PD-L1) actually made the alarm bells ring louder in the factory. But adding the Tasquinimod "mute button" silenced those bells, allowing the treatment to work better.

5. Why This Matters for Humans

The researchers didn't just look at mice; they checked rib bones from human lung cancer patients. They found the same "alarm bells" (S100A9 and LCN2) ringing in the human factories too.

The Big Takeaway:
Lung cancer is a systemic disease—it doesn't just live in the lungs; it hijacks the body's entire immune factory. By targeting the "Master Alarm" (S100A9) in the bone marrow, we might be able to stop the cancer from recruiting its own army and help the body's natural defenses fight back, especially for patients who aren't responding to current treatments.

In a nutshell: Cancer turns the body's immune factory into a chaotic mess of raw materials. This study found the specific "alarm" causing the chaos and showed that silencing it can shrink the tumor and help the body heal.

Technical Summary

1. Problem Statement

Lung cancer, particularly Non-Small Cell Lung Cancer (NSCLC), is a leading cause of cancer-related death. A major clinical challenge is the high rate of primary or secondary resistance to immunotherapy (IT), specifically immune checkpoint inhibitors (ICIs) targeting PD-1/PD-L1. This resistance is often linked to the infiltration of immunosuppressive myeloid cells within the tumor microenvironment (TME).

While it is known that these myeloid cells originate from the bone marrow (BM) via "emergency myelopoiesis," the specific molecular mechanisms driving the remote alteration of the BM niche by lung tumors remain poorly understood. Key gaps include:

• The spatiotemporal complexity of hematopoiesis makes the BM difficult to access and study.
• The specific transcriptional and proteomic changes in hematopoietic stem and progenitor cells (HSPCs) during lung cancer progression are not fully characterized.
• The role of specific biomarkers (like S100A9 and LCN2) in regulating this process and their potential as therapeutic targets is unclear.

2. Methodology

The study employed a multi-modal approach integrating murine models and human clinical samples to characterize the BM niche in lung cancer.

• Animal Model: Orthotopic Lewis Lung Carcinoma (LLC) models were established in C57BL/6J mice (both intravenous and intrathoracic injection methods). Age-matched healthy controls were used for comparison.
• Single-Cell RNA Sequencing (scRNA-seq): Hashed scRNA-seq was performed on flushed BM cells from LLC-bearing mice and controls. Data was processed using Seurat (integration, clustering, annotation) and Monocle3 (trajectory analysis).
• Spatial and Functional Validation:
  • Flow Cytometry: Used to validate cell population frequencies (HSCs, MPPs, GMPs, etc.) and protein expression (S100A9, LCN2, c-Kit) in BM and spleen.
  • Two-Photon Microscopy: Intravital imaging of the calvarium BM was used to visualize HSPC homing, vascular localization, and c-Kit expression in live mice.
  • Immunohistochemistry (IHC): Performed on fixed murine humeri and human rib fragments to assess spatial protein distribution.
• Molecular Analysis:
  • RT-qPCR: Quantified gene expression (S100A9, LCN2, CXCL12, Il17a) in crushed BM, blood, and human rib fragments.
  • ELISA: Measured secreted levels of S100A9, LCN2, and IL-17A in serum, BM supernatants, and lung tissue.
  • In Silico Analysis: NicheNet was used to predict ligand-receptor interactions and upstream regulators.
• Therapeutic Intervention: An in vivo survival and tumor growth study was conducted treating LLC-bearing mice with:
  • Anti-PD-L1 monoclonal antibody (Immunotherapy).
  • Tasquinimod (an oral S100A9 inhibitor).
  • Combination therapy.
• Human Samples: Rib fragments from operable NSCLC patients (Stages I-II) and healthy donors were analyzed via RT-qPCR and IHC.

3. Key Contributions

• Systemic Characterization of the BM Niche: Provided the first comprehensive single-cell resolution map of the hematopoietic compartment in the BM of lung cancer-bearing subjects, revealing a systemic shift toward emergency myelopoiesis.
• Identification of S100A9 and LCN2 as Key Drivers: Discovered that S100A9 and Lipocalin-2 (LCN2) are significantly upregulated across the entire hematopoietic trajectory in lung cancer, acting as potential upstream regulators of the immunosuppressive milieu.
• Mechanistic Insight into Differentiation Arrest: Demonstrated that the accumulation of HSPCs in the BM is driven by a differentiation arrest rather than increased proliferation, coupled with altered mobilization dynamics (downregulation of CXCL12/CXCR4 axis).
• Therapeutic Validation: Showed that inhibiting S100A9 with Tasquinimod reduces tumor growth, even in the context of immunotherapy, by altering the secretion profile of S100A9 and LCN2 in the BM.

4. Key Results

A. Hematopoietic Shifts in the Bone Marrow

• Accumulation of Progenitors: LLC-bearing mice exhibited a significant increase in Hematopoietic Stem Cells (HSCs), MultiPotent Progenitors (MPPs), Granulocyte Monocyte Progenitors (GMPs), and early Granulocyte Progenitors (GPs).
• Differentiation Arrest: This accumulation was attributed to a block in differentiation rather than hyper-proliferation (no upregulation of Mki67 in HSPCs).
• Lineage Skewing: There was a predominance of myeloid and lymphoid progenitors at the expense of mature neutrophils and B cells.
• Mobilization Defect: Despite increased mobilization signals, HSPCs remained in the BM due to a downregulation of the retention factor CXCL12 in the stromal niche and its receptor CXCR4 on hematopoietic cells, suggesting a complex dysregulation of the CXCL12-CXCR4 axis.

B. Upregulation of S100A9 and LCN2

• Transcriptional & Protein Levels: S100a9 and Lcn2 were consistently upregulated across almost all hematopoietic clusters (HP, MoDC, Ery, B-cell, etc.) in LLC mice.
• Secretion Dynamics:
  • S100A9: Detected in BM supernatants but not serum or lung TME, suggesting paracrine action within the BM.
  • LCN2: Markedly increased in blood and BM supernatants but reduced in the lung TME.
• Regulatory Relationship: NicheNet analysis and in vivo data suggest S100A9 acts as an upstream regulator of LCN2.
• Independence from IL-17A: Contrary to previous literature, the upregulation of S100A9 and LCN2 in this model was not driven by IL-17A, as Il17a expression remained unchanged.

C. Therapeutic Effects of Tasquinimod

• Tumor Reduction: Treatment with Tasquinimod (S100A9 inhibitor) significantly reduced lung tumor volume and weight, both as monotherapy and in combination with anti-PD-L1.
• Mechanism of Action:
  • Tasquinimod treatment led to a reduction in secreted S100A9 and LCN2 in the BM supernatant.
  • It appeared to reverse the differentiation arrest, shifting progenitors toward mature cells (increased Ly6G+ neutrophils, decreased GMPs).
  • It counteracted the effect of anti-PD-L1, which alone increased the secretion of S100A9 and LCN2 in the BM.

D. Validation in Human NSCLC

• Patient Samples: Analysis of rib fragments from NSCLC patients confirmed the upregulation of S100A9 (79% of patients) and LCN2 (57% of patients) compared to healthy controls.
• Stromal Alteration: Human samples also showed downregulation of CXCL12, mirroring the murine findings and confirming the relevance of the BM niche alteration in human disease.

5. Significance

• Novel Biomarkers: Identifies S100A9 and LCN2 as critical biomarkers for the systemic impact of lung cancer on the bone marrow, potentially useful for predicting immunotherapy resistance.
• Therapeutic Target: Proposes S100A9 inhibition (via Tasquinimod) as a viable strategy to treat immunotherapy-refractory lung cancer by resetting the hematopoietic niche and reducing the pool of immunosuppressive myeloid cells.
• Paradigm Shift: Challenges the view that emergency myelopoiesis is solely driven by IL-17A, highlighting a distinct S100A9-LCN2 axis.
• Clinical Relevance: The findings bridge the gap between preclinical mouse models and human NSCLC, suggesting that targeting the bone marrow niche could improve outcomes for patients with advanced lung cancer who fail standard immunotherapy.