An immunomechanical checkpoint PYK2 governs monocyte-to-macrophage differentiation in pancreatic cancer

Background

A widespread feature of cancer development is the stiffening of the surrounding microenvironment caused by matrix deposition by fibroblasts and crosslinking by macrophages (Tharp et al. 2024). Matrix rigidity has been shown to exclude immune cells from the tumor mass and facilitate immune suppression and cancer metastasis. Pancreatic ductal adenocarcinoma (PDAC) is composed of a dense desmoplastic stroma and characterized by high infiltration of immunosuppressive macrophages (Bräutigam et al. 2025). While biochemical factors that induce monocyte differentiation into immunosuppressive macrophages are quite well studied, the mechanisms of how mechanical stimuli influence this maturation process are largely unknown (Jakubzick et al. 2017; Taufalele et al. 2023).

In this preprint, Xie and colleagues found that the FAK component PYK2 is important to instruct monocytes-to-macrophages differentiation by integrating mechanical cues and transcriptional regulation and that interfering with PYK2 signaling disrupts monocyte-to-immunosuppressive macrophage polarization and enhances immunotherapy efficacy in PDAC.

Key findings of this preprint

1. EMT mechanics promotes macrophage differentiation in PDAC

Using Masson’s trichrome staining, the authors first confirmed that PDAC shows abundant matrix deposition and fibrotic area. By integrating spatial transcriptomics and external single cell data with measurement of tissue stiffness, the authors observed a correlation between tumor rigidity, fibroblasts, macrophages and gene expression profiles related to mechanical modules (Fig.1).

To study the contribution of tissue mechanics to monocyte-to-macrophage differentiation, the authors manipulated tumor stiffness in a mice model of PDAC by orthotopically implanting cancer (KPC) cells mixed either with soft or stiff hydrogels. Notably, cancer cells mixed with stiff hydrogels showed increased infiltration of macrophages. Consistently, in vitro differentiation of macrophages demonstrated that even bone marrow-derived macrophages are sensitive to mechanical cues upon maturation, with progenitor cells cultured on soft matrices displaying a monocyte-like phenotype and cells cultured on stiff matrices showing a more macrophage-skewed differentiation (Fig.1).

2. PYK2 is upregulated by mechanical stimulation in monocytes

By integrating publicly available scRNA-seq datasets of blood and tumor cells in PDAC patients, the authors deconvoluted three states of monocytes differentiation into macrophages (Fig.2). Cells in state 2, namely monocytes that infiltrated PDAC, showed an initial mechanical sensing signature, with upregulation of cytoskeletal genes, including PYK2. Importantly, PYK2+ cells were observed to reside in fibrotic stiff regions in both human and murine PDAC, suggesting a mechanosensitive role for PYK2 in monocyte to macrophage maturation (Fig.2).

3. PYK2 is mechanoresponsive and instructs monocytes differentiation

To validate PYK2 as a mechanoresponsive gene, THP-1 cells and bone marrow progenitors were cultured on substrates or left without adhesion during differentiation into macrophages. Meanwhile, PYK2 levels and phosphorylation were monitored by western blot. PYK2 phosphorylation was also monitored in macrophages in vivo by tumor-matrix implantation (as previously described) and by conditionally knocking-out PYK2 in macrophages. Altogether, this set of experiments showed that phosphorylation of PYK2 and acquisition of immunomodulatory markers increased in macrophages from stiffer tumors and that these features were lost in PYK2 KO macrophages (Fig.3).

To further elucidate the mechanism of PYK2 activation, which is known to be Ca²⁺ dependent, the authors evaluated the role of Piezo1, a well-known mechanoresponsive ion channel. Indeed, PYK2 activation was observed to rely on Piezo1 Ca²⁺ influx and Arp2/3-mediated actin polymerization. Importantly, activated PYK2 was shown to translocate into the nucleus of maturating monocytes and participate in the transcription of mechanical-responsive genes and genes related to macrophage differentiation.

4. PYK2 KO sensitizes PDAC to PD-1 therapy

To evaluate whether PYK2 KO in monocytes could be beneficial in reducing tumor development, the author injected orthotopic tumors in mice lacking PYK2 specifically in monocytes. Although diminished fibrosis and tumor size were observed at early stages of tumorigenesis, at late stages these differences were much smaller, potentially indicating some mechanisms adopted by tumors to circumvent the pro-tumorigenic role of immunomodulatory macrophages. Despite this, CD8 cytotoxic T cell were more abundant and effective in tumors from PYK2-deficient mice and PD-1 blockade increased CD8 T cell antitumor immunity, synergizing with the beneficial depletion of immunomodulatory macrophages and extending the life of these mice.

In summary, this study highlights the relevance and potential clinical implication of targeting myeloid mechanosensing in combinatory therapies in PDAC, to overcome immune dysfunction and sensitize this highly aggressive tumor to immunotherapies.

Why I highlight this study

The huge amount of data presented in this preprint is of high value not only to cancer biologists, but also to cell biologists and clinicians. Considering cancer as a heterogeneous disease not only from a genetic point of view, but also integrating physical and mechanical properties of the surrounding tumor microenvironment, are crucial for better understanding each type of cancer and finding potential vulnerabilities to target in combinatorial therapies.

Questions for the authors

• Do you have any hypothesis explaining why, at late stages, tumors reach comparable sizes even in mice bearing PYK2-deficient monocytes?
• How do you reconcile the observation that CK666 is not effective in inhibiting Arp2/3 in BMDM (Cao et al. 2024), with your result showing complete abrogation of BMDM differentiation in the presence of CK666?

References

Tharp KM, Kersten K, Maller O, Timblin GA, Stashko C, Canale FP, et al. Tumorassociated macrophages restrict CD8+ T cell function through collagen deposition and metabolic reprogramming of the breast cancer microenvironment. Nat Cancer. 2024;5:1045–62.

Bräutigam, K et al. Tumor Immune Microenvironment in Pancreatic Ductal Adenocarcinoma revisited – Exploring the “Space”. Cancer Letters. 2025 doi:10.1016/J.CANLET.2025.217699

Jakubzick C V., Randolph GJ, Henson PM. Monocyte differentiation and antigen presenting functions. Nat Rev Immunol. 2017;17:349–62.

Taufalele P V., Wang W, Simmons AJ, Southard-Smith AN, Chen B, Greenlee JD, et al. Matrix stiffness enhances cancer-macrophage interactions and M2-like macrophage accumulation in the breast tumor microenvironment. Acta Biomater. 2023;163:365–77

Cao et al. CK-666 and CK-869 differentially inhibit Arp2/3 iso-complexes. EMBO Reports. 2024; https://doi.org/10.1038/s44319-024-00201-x

Tags: cell mechanics, immunosuppression, macrophages, pancreatic cancer, pyk2

doi: https://doi.org/10.1242/prelights.40442

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