Single-cell RNA-seq reveals cancer pain: a subset of neuron-like macrophages associated with cancer pain

Background cancers
remain the leading cause of
death worldwide.
Many patients with malignant tumors endure chronic pain, especially those with advanced lung, breast, prostate or gastrointestinal cancer, which seriously affects their quality of life
.
Elucidating the underlying mechanisms of cancer pain is important
to guide the development of treatment.
 

Cancer pain is thought to be caused in part by tissue damage and inflammation in the tumor microenvironment (TME), but the mechanism is unclear
.
Chronic inflammation is often associated with the development and progression of cancer, and it is unclear how chronic inflammation drives cancer complications – especially the onset
of chronic pain.
Neuronal plasticity has long been recognized as a key mechanism for
the development and maintenance of chronic pain.
Peripheral sensitization at the nociceptor level, usually after peripheral tissue injury and inflammation, is key
to transforming acute injury sensations into chronic pain.
Nociceptive sensory neurons not only respond to immune signals, but also directly modulate the inflammatory response – silencing nociceptors can reduce inflammation in allergic airways as proof of this (nerve cells are involved in immunity).

There is growing evidence that many non-neuronal cell types, including macrophages, play an important role
in pain perception.
A novel role
of macrophages in peripheral pain regulation has been proposed.
Depletion of macrophages in mice, rather than neutrophils or T cells, effectively reduces the development of incisions and mechanical and thermal sensitivity caused by pathogens, accompanied by downregulation
of interleukin-1β (IL-1β) and other analgesic mediators at the site of inflammation.
Macrophages have also been found to affect analgesia
by indirectly releasing anti-inflammatory mediators such as IL-10 and specialized prolytic mediators.
(Immune cells are involved in pain sensation).

Research highlights

Researchers at Chinese University of Hong Kong successfully used single-cell RNA sequencing to analyze the dynamic changes of macrophages in the transcriptome under tissue inflammation and cancer conditions, discovered a phenomenon of "macrophage to neuron-like cell transition" (MNT) that directly promotes tumor neurogenesis, and conducted macrophage depletion and fate localization studies in lung cancer models.
Macrophage-specific Smad3 was identified as a key regulator of promoting MNT at the genomic level; Its destruction effectively blocks the innervation of the tumor and tumor-dependent nociceptive behavior
in the body.
Therefore, "macrophage-neuron-like cell transformation" (MNT) may be a precise therapeutic target for cancer pain
.

1.
The authors used flow cytometry to isolate cells of macrophage lineage from tumors in the LysM-Cre/ROSA-tdTomato mouse LLC lung cancer model for 10× Genomics scRNA-seq analysis—single-cell RNA sequencing (scRNA-seq) is an emerging method for resolving cellular heterogeneity at the genome-wide scale It can be used to distinguish transcription network timing during development or external stimulation, which is often obscured in population analysis; The transcriptome characteristics and phenotypes in the tumor microenvironment are highly dynamic – the phenotype of macrophages can be rapidly altered
by transcriptional regulation due to stimulation by cancer cells.
They found a group of tumor-associated macrophages (TAMs) highly expressing the neuronal differentiation marker Tubb3, whose upregulated differentially expressed genes were highly correlated
with neurogenesis, according to GO analysis on the MetaCore bioinformatics platform.
The researchers found that the transcriptional level of Tubb3⁺ TAM cells of such LLC tumors in vivo showed a loss of macrophage marker expression, while neurogenesis-related genes such as Map1b, Syt13, Cyb5d2, Pax6, Cdon, Wnt5a, Pex2, etc.
were expressed
.
Two-photon in vivo imaging further confirms the presence of this macrophage lineage (tdTomato+)-derived cell with a neurofibroid morphology with enhanced
calcium efflux levels (OGB-1+).
Liposomal clodronate-mediated macrophage depletion eliminates the formation of this neuron-like subset in LLC tumors, and the presence of Tubb3⁺ TAM cells is significantly associated with cancer-associated
pain response behavior.

The authors also found a subset of TUBB3⁺CD68 TAM (with strong expression of neuronal genes including BMP7, SHANK, CHL1, and PAX6) in 10× Genomics
' scRNA-seq human non-small cell lung cancer (NSCLC) biopsy dataset.
This previously unknown subset of TUBB3+ TAM exhibits neuronal-like transcriptome characteristics similar to TUBB3⁺ neuronal cells and is highly correlated
with human neurological diseases.
The presence of this neuron-like TAM subset was also found in human renal cell carcinoma and hepatocellular carcinoma and further confirmed
in large-scale biopsies of NSCLC patients.
Visualization of TAM subsets in NSCLC by confocal 3D imaging and detection of tumor-specific localization by flow cytometry analysis, comparing NSCLC and normal lung tissue showed that TUBB3⁺ TAM subtype was highly present in adenocarcinoma NSCLC and positively correlated with
the expression of neuronal differentiation marker TUBB3 in human TME.
Notably, the abundance of TUBB3+ TAMs (TUBB3+ CD68+/CD68⁺ ratio > 30%) was significantly associated with poorer survival in older NSCLC patients, and their upregulated differential-expression genes were highly associated with neurological disorders (Huntington's disease, Parkinson's disease, and Alzheimer's disease).
Indicates the potential contribution
of this previously unknown subset of tumor-associated macrophages to neurogenesis.

To better understand the dynamics and potential contributions of Tubb3+ TAM subsets in tumor neurogenesis, the authors analyzed Tubb3⁺ TAM data using the latest bioinformatics platform RNA rate analysis, a dynamic model of RNA transcription and cleavage for evaluating individual cells for causal inference between final differentiated cells and parental cells.
Both methods (RNA rate and MuTrans assay) confirmed that Tubb3⁺ TAM subsets were derived from macrophage lineages
.
Confocal imaging techniques observed macrophage line-derived Tubb3+ cells in live LysM-Cre/ROSA-tdTomato mouse LLC tumors, accounting for more than
90% of the total number of tumor Tubb3⁺ cells detected by flow cytometry and image analysis 。 In order to confirm the possibility of macrophages directly transforming into neuron-like cells in TME, the results confirmed the complete transformation of Tubb3−Cd68+ TAM into Tubb3⁺ Cd68⁻Neuron-like cells
at the transcriptional level by analyzing the relationship between their developmental stages in the tumor microenvironment 。 These findings suggest the previously unknown phenomenon of "macrophage to neuronal-like cell transformation" (MNT), which may be a previously unknown source of abundant neuron-like cells that directly promotes tumor innervation
.

3.
In order to better understand the role of Tubb3+ TAM subsets in tumor neurogenesis, the authors performed macrophage depletion in LysM-Cre/iDTR mice in the LLC homologous lung cancer model, and found that Tubb3+ TAMs were significantly reduced, and Tubb3⁺ TAMs in LLC tumors could be successfully reversed through myeloid macrophage (BMDM) over-metastasis in vivo
。 They also found that in vitro LLC cancer cell-conditioned medium (CM) stimulated neuronal morphology in BMDM; BMDM was found to have fibroblast-like morphology
under TGF-β1 stimulation.
TGF-β1 pretreatment can further enhance cancer cell culture media-induced BMDM neuronal morphology
.
Higher expression of neuronal differentiation mediators (i.
e.
, Tubb3, NeuN, and Pou4f1) was detected in CM-stimulated BMDM compared to in vitro TGF-β1, indicating that cancer conditions are a key factor
in initiating "macrophage to neuron-like cell transformation" (MNT).

Unbiased bioinformatics analysis of differentially expressed genes upregulated by MNTs revealed gene networks
associated with neurotransmitter release and receptor expression.
Macrophage lineage scRNA-seq analysis showed that the expression of presynaptic membrane calcium ion sensors (Syt13 and Syt14), neurotransmitter synthase (Aldh9a1 and Dagla), and sensory neuron functional markers (TrkA and Prph) was highly enriched in "macrophage to neuron-like cell transformation" (MNT), suggesting a potential role that MNT may play in tumor sensation and may lead to cancer pain
.

Using the classical nociceptive stimulation capsaicin (an agonist of the TRPV protein responsible for responding to the sensation of cancer pain) to characterize the biological function of MNTs by imaging calcium efflux by OGB-1, it was found that BMDM-derived MNTs (Tubb3+ tdTomato+ cells) had a calcium flow response, while no response
was detected in unstimulated BMDM controls in vitro.
BMDM-derived MNTs also respond to
stimulation by GSK1016790A (pain receptor Trpv4 agonist) and KCl (neuronal voltage-gated calcium channel agonist).
(Immune cells under specific conditions are involved in neural responses)

Local adoptive transfer of MNTs generated in vitro to non-obese diabetes/severe combined immunodeficiency (NOD/SCID) mice with macrophage dysfunction significantly increased cancer-associated nociceptive responses
in NOD/SCID mice 。 Activation of neuronal calcium signals (p-CREB and Camk2d) was detected in both human and mouse MNTs.
In particular, the detection of specific expression of pain receptors Ano1 and Trpc1 in MNTs, as well as the link between MNTs expressing synaptophysin in vivo LLC tumors and neural circuit formation of BMDM-derived MNTs in vitro, demonstrated the involvement of MNTs in TME in promoting cancer pain
.
These data suggest that MNTs may exhibit nociceptor-receptor-like activity in tumors, which may be a potential therapeutic target for
cancer pain.

4.
Confocal microscopy confirmed the presence of overactivation of SMAD3 in MNTs from human NSCLC biopsy samples, and Opal multiplex immunohistochemical staining confirmed the relationship between
macrophage-specific Smad3 and the abundance of MNTs in human NSCLC.
Therefore, the authors investigated the regulatory role
of Smad3-dependent TME in MNT in an experimental lung cancer model.
It was found that in LLC tumor TME with Smad3 knockout (Smad3-KO mice), not only MNT but also tumor-related nociceptive behavior was greatly inhibited, which means that Smad3-dependent TME plays a crucial role
in MNT regulation.

In vitro experiments, Smad3 deletion significantly prevented LLC cancer cell medium from inducing wild-type BMDMs to express neuronal
markers.
By transferring Smad3-wild-type (WT) or Smad3-KO type BMDMs into a NOD/SCID mouse LLC lung cancer model with macrophage dysfunction, the authors found that adoptive metastases wild-type BMDM significantly increased MNT and cancer-associated nociceptive behaviors, while adoptive metastases lacked BMDMs (KO-BMDM) significantly inhibited MNT, suggesting the importance of
Smad3 in MNT.

Drug inhibition of Smad3 with the specific inhibitor SIS3 can effectively block "macrophage to neuron-like cell transformation" (MNT) and its associated tumor neural distribution (Tubb3+ and NeuN⁺ cells) and cancer-associated spontaneous injury behavior
in LLC-carrying mice.
Thus, Smad3 may be a drug target targeting MNT-driven cancer neurogenesis
.