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Updated: Aug 15, 2025
In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window
Published on: August 4, 2022
In vivo morphological alterations of TAMs during KCa3.1 inhibition-by using in vivo two-photon time-lapse technology
Francesca Massenzio1, Marco Cambiaghi1, Federica Marchiotto1
1Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Verona, Italy.
Abstract:
Tumor associated macrophages (TAMs) are the mostprevalent cells recruited in the tumor microenvironment (TME). Once recruited, TAMs acquire a pro-tumor phenotype characterized by a typical morphology: ameboid in the tumor core and with larger soma and thick branches in the tumor periphery. Targeting TAMs by reverting them to an anti-tumor phenotype is a promising strategy for cancer immunotherapy. Taking advantage of Cx3cr1GFP/WT heterozygous mice implanted with murine glioma GL261-RFP cells we investigated the role of Ca2+-activated K+ channel (KCa3.1) on the phenotypic shift of TAMs at the late stage of glioma growth through in vivo two-photon imaging. We demonstrated that TAMs respond promptly to KCa3.1 inhibition using a selective inhibitor of the channel (TRAM-34) in a time-dependent manner by boosting ramified projections attributable to a less hypertrophic phenotype in the tumor core. We also revealed a selective effect of drug treatment by reducing both glioma cells and TAMs in the tumor core with no interference with surrounding cells. Taken together, our data indicate a TRAM-34-dependent progressive morphological transformation of TAMs toward a ramified and anti-tumor phenotype, suggesting that the timing of KCa3.1 inhibition is a key point to allow beneficial effects on TAMs.
Insights
Targeting tumor-associated macrophages (TAMs) with KCa3.1 inhibitors like TRAM-34 promotes a shift toward an anti-tumor phenotype. This morphological transformation in the tumor core suggests a promising strategy for glioma immunotherapy.
Area of Science:
- Immunology
- Cancer Biology
- Neuro-oncology
Background:
- Tumor-associated macrophages (TAMs) are key regulators of the tumor microenvironment (TME).
- TAMs typically adopt a pro-tumor phenotype, characterized by distinct morphologies in the tumor core and periphery.
- Reversing TAM phenotype is a promising cancer immunotherapy strategy.
Purpose of the Study:
- To investigate the role of the Ca2+-activated K+ channel (KCa3.1) in TAM phenotypic shifts during late-stage glioma growth.
- To evaluate the effect of KCa3.1 inhibition on TAM morphology and function in vivo.
Main Methods:
- Utilized Cx3cr1GFP/WT heterozygous mice bearing GL261-RFP glioma cells.
- Employed in vivo two-photon imaging to observe TAMs.
- Administered a selective KCa3.1 inhibitor (TRAM-34) to assess its effects.
Main Results:
- KCa3.1 inhibition by TRAM-34 induced a time-dependent shift in TAM morphology, increasing ramified projections.
- TAMs exhibited a less hypertrophic phenotype in the tumor core following TRAM-34 treatment.
- TRAM-34 selectively reduced glioma cells and TAMs in the tumor core without affecting surrounding cells.
Conclusions:
- TRAM-34 treatment drives a progressive morphological transformation of TAMs towards a ramified, anti-tumor phenotype.
- The timing of KCa3.1 inhibition is critical for achieving beneficial effects on TAMs in glioma.
- Targeting KCa3.1 offers a potential therapeutic strategy for modulating TAMs in cancer immunotherapy.
