Immune reprogramming of 3D tumor models via optoporation-mediated targeted gene delivery to macrophages

Abstract

The dynamics of the tumor microenvironment (TME) are a key determinant of cancer progres-sion and therapeutic resistance through complex interactions between tumor, stromal and im-mune cell populations. Among these, tumor-associated macrophages (TAMs) play a central role in promoting tumor growth and immune suppression. However, the specific contributions of TAMs remain poorly understood due to the lack of tools enabling selective genetic manipulation in three-dimensional (3D) tumor models. Here, we present a gold nanoparticle-assisted opto-poration approach that enables spatially selective plasmid-based gene delivery to TAMs within intact heterocellular 3D pancreatic ductal adenocarcinoma (PDAC) spheroids, thereby modulat-ing the TME. In two-dimensional (2D) TAM cultures, conventional transfection of IRF5- and IKBKB-encoding plasmids validated their capacity to induce TAM repolarization, as evidenced by activation of interferon signaling. Extending this approach to 3D PDAC spheroids, nanopar-ticle-assisted optoporation achieved selective transfection of TAMs with IRF5- and IKBKB-encoding plasmids by transiently generating nanoscale membrane pores in illuminated cells. TAMs transfection elicited a robust interferon response, marked by transcriptional upregulation of IFNA, IFNB1, and CXCL10, and increased protein levels of IFNB1, IFNL1, and CXCL13, together with downregulation of pro-tumorigenic markers CEACAM5, IL19, and IL32. These coordinated changes indicate a shift towards an anti-tumorigenic TME. By enabling minimally invasive, TAM-specific gene delivery in complex multicellular 3D spheroids, this strategy al-lows precise modulation of the TME and opens new avenues for modeling its dynamics in can-cer progression and therapeutic response.

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BibTeX RIS

BibTeX

@misc{poljak2026immune,
  title = {{Immune reprogramming of 3D tumor models via optoporation-mediated targeted gene delivery to macrophages}},
  author = {Ines Poljak and Izzah Nabilah Hussein and Chenlei Gu and Giulio Giustarini and Teng Xiang and Yusuke Toyama and Ciro Chiappini and Giulia Adriani},
  howpublished = {BioRxiv},
  year = {2026},
  month = oct,
  doi = {10.64898/2026.03.16.712123},
  url = {https://hal.science/hal-05776458v1},
  note = {CC BY},
}

RIS

TY  - UNPB
TI  - Immune reprogramming of 3D tumor models via optoporation-mediated targeted gene delivery to macrophages
AU  - Ines Poljak
AU  - Izzah Nabilah Hussein
AU  - Chenlei Gu
AU  - Giulio Giustarini
AU  - Teng Xiang
AU  - Yusuke Toyama
AU  - Ciro Chiappini
AU  - Giulia Adriani
PY  - 2026
DA  - 2026/10/03
PB  - BioRxiv
DO  - 10.64898/2026.03.16.712123
UR  - https://hal.science/hal-05776458v1
AB  - The dynamics of the tumor microenvironment (TME) are a key determinant of cancer progres-sion and therapeutic resistance through complex interactions between tumor, stromal and im-mune cell populations. Among these, tumor-associated macrophages (TAMs) play a central role in promoting tumor growth and immune suppression. However, the specific contributions of TAMs remain poorly understood due to the lack of tools enabling selective genetic manipulation in three-dimensional (3D) tumor models. Here, we present a gold nanoparticle-assisted opto-poration approach that enables spatially selective plasmid-based gene delivery to TAMs within intact heterocellular 3D pancreatic ductal adenocarcinoma (PDAC) spheroids, thereby modulat-ing the TME. In two-dimensional (2D) TAM cultures, conventional transfection of IRF5- and IKBKB-encoding plasmids validated their capacity to induce TAM repolarization, as evidenced by activation of interferon signaling. Extending this approach to 3D PDAC spheroids, nanopar-ticle-assisted optoporation achieved selective transfection of TAMs with IRF5- and IKBKB-encoding plasmids by transiently generating nanoscale membrane pores in illuminated cells. TAMs transfection elicited a robust interferon response, marked by transcriptional upregulation of IFNA, IFNB1, and CXCL10, and increased protein levels of IFNB1, IFNL1, and CXCL13, together with downregulation of pro-tumorigenic markers CEACAM5, IL19, and IL32. These coordinated changes indicate a shift towards an anti-tumorigenic TME. By enabling minimally invasive, TAM-specific gene delivery in complex multicellular 3D spheroids, this strategy al-lows precise modulation of the TME and opens new avenues for modeling its dynamics in can-cer progression and therapeutic response.
SN  - 2692-8205
ER  -

Abstract

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