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Assessing efficacy and immune-stimulatory effects of tumor-derived dendritic cell reprogramming using immuno-competent 3D tumor spheroid model

Agarkova, Irina ; Rotankova, Nadezhda ; Rudnik, Michal ; Strebel, Silvan ; Zimmermannova, Olga LU ; Rosa, André LU ; Fiúza Rosa, Fábio LU ; Moritz, Wolfgang ; Pereira, Carlos-Filipe LU orcid and Pires, Cristiana LU (2023) American Association for Cancer Research Annual Meeting 2023 In Cancer Research 83(7 Supplement).
Abstract
Immunotherapy has brought hope for cancer treatment, but its clinical success remains limited. Recently, overexpression of the transcription factors PU.1, IRF8 and BATF3 (PIB) was shown to induce direct reprogramming of tumor cells into antigen-presenting type 1 conventional dendritic cells (cDC1s), a rare subset of immune cells with pivotal role in anti-cancer immunity. This strategy might open avenues to enhance cancer cell recognition and elimination by the immune system. However, currently existing in-vitro and in-vivo testing platforms do not qualify to reproduce all complex cell interactions essential for the approbation of this hypothesis. Here, we report the development of the InSphero 3D InSight™ Oncology Platform for in-vitro... (More)
Immunotherapy has brought hope for cancer treatment, but its clinical success remains limited. Recently, overexpression of the transcription factors PU.1, IRF8 and BATF3 (PIB) was shown to induce direct reprogramming of tumor cells into antigen-presenting type 1 conventional dendritic cells (cDC1s), a rare subset of immune cells with pivotal role in anti-cancer immunity. This strategy might open avenues to enhance cancer cell recognition and elimination by the immune system. However, currently existing in-vitro and in-vivo testing platforms do not qualify to reproduce all complex cell interactions essential for the approbation of this hypothesis. Here, we report the development of the InSphero 3D InSight™ Oncology Platform for in-vitro assessment of efficacy and immune-stimulatory effects of this novel cancer immunotherapy approach. The feasibility of 3D spheroid formation for several GFP-expressing tumor cell lines was evaluated by varying seeding conditions in AKURA 96 well plate. We have measured the growth (ATP content) and GFP signal overtime and analyzed the morphology of the spheroids by IHC. With this, we have established spheroid models of T98G (glioblastoma), PK59 (pancreatic cancer), and A375 (melanoma) cell lines that are growing and viable for at least 10 days. In parallel, using 2D cultures, we have identified the optimal multiplicity of infection of a lentiviral vector encoding for PIB and mCherry to enable high transduction (mCherry+ cells), reprogramming efficiency (mCherry+CD45+HLA-DR+ cells), and cell viability, quantified by flow cytometry and IHC. Then, we have demonstrated that cDC1 reprogramming progresses in the context of 3D cancer spheroids and tumor cells acquire expression of CD45+ and HLA-DR+ cells using IHC and confocal microscopy analysis. We developed an algorithm enabling automated analysis of confocal images and quantification of cDC1 reprogramming efficiency from individual image stacks calculated as a ratio of mCherry+, CD45+ and HLA-DR+ cells versus the number of DAPI+ nuclei. Using the new algorithm we have evaluated the reprogramming efficacy of the different virus dosages in all three types of 3D tumor spheroids. Lastly, we have cocultured tumor spheroids transduced with PIB with naïve or activated HLA-matched PBMCs and evaluated cytokine secretion as a readout of immune cell activation. We observed that reprogramming induces activation of T cells and correlated it to the number of reprogrammed cells in the tumor spheroid, evaluated by the HC imaging. In summary, we developed the InSphero 3D InSight™ Oncology Platform that allowed us to demonstrate the effects of direct reprogramming of tumor cells into immunogenic dendritic cells. Combined with high-content imaging analysis, this platform offers a powerful solution for preclinical translational research. (Less)
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author
; ; ; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Cancer Research
volume
83
issue
7 Supplement
article number
4550
pages
1 pages
publisher
American Association for Cancer Research Inc.
conference name
American Association for Cancer Research Annual Meeting 2023
conference dates
2023-04-14 - 2023-04-19
ISSN
1538-7445
DOI
10.1158/1538-7445.AM2023-4550
language
English
LU publication?
yes
id
50d2ed2c-5f58-48b7-9cef-fed2fabc2249
date added to LUP
2023-08-10 20:57:07
date last changed
2023-08-11 09:44:16
@misc{50d2ed2c-5f58-48b7-9cef-fed2fabc2249,
  abstract     = {{Immunotherapy has brought hope for cancer treatment, but its clinical success remains limited. Recently, overexpression of the transcription factors PU.1, IRF8 and BATF3 (PIB) was shown to induce direct reprogramming of tumor cells into antigen-presenting type 1 conventional dendritic cells (cDC1s), a rare subset of immune cells with pivotal role in anti-cancer immunity. This strategy might open avenues to enhance cancer cell recognition and elimination by the immune system. However, currently existing in-vitro and in-vivo testing platforms do not qualify to reproduce all complex cell interactions essential for the approbation of this hypothesis. Here, we report the development of the InSphero 3D InSight™ Oncology Platform for in-vitro assessment of efficacy and immune-stimulatory effects of this novel cancer immunotherapy approach. The feasibility of 3D spheroid formation for several GFP-expressing tumor cell lines was evaluated by varying seeding conditions in AKURA 96 well plate. We have measured the growth (ATP content) and GFP signal overtime and analyzed the morphology of the spheroids by IHC. With this, we have established spheroid models of T98G (glioblastoma), PK59 (pancreatic cancer), and A375 (melanoma) cell lines that are growing and viable for at least 10 days. In parallel, using 2D cultures, we have identified the optimal multiplicity of infection of a lentiviral vector encoding for PIB and mCherry to enable high transduction (mCherry+ cells), reprogramming efficiency (mCherry+CD45+HLA-DR+ cells), and cell viability, quantified by flow cytometry and IHC. Then, we have demonstrated that cDC1 reprogramming progresses in the context of 3D cancer spheroids and tumor cells acquire expression of CD45+ and HLA-DR+ cells using IHC and confocal microscopy analysis. We developed an algorithm enabling automated analysis of confocal images and quantification of cDC1 reprogramming efficiency from individual image stacks calculated as a ratio of mCherry+, CD45+ and HLA-DR+ cells versus the number of DAPI+ nuclei. Using the new algorithm we have evaluated the reprogramming efficacy of the different virus dosages in all three types of 3D tumor spheroids. Lastly, we have cocultured tumor spheroids transduced with PIB with naïve or activated HLA-matched PBMCs and evaluated cytokine secretion as a readout of immune cell activation. We observed that reprogramming induces activation of T cells and correlated it to the number of reprogrammed cells in the tumor spheroid, evaluated by the HC imaging. In summary, we developed the InSphero 3D InSight™ Oncology Platform that allowed us to demonstrate the effects of direct reprogramming of tumor cells into immunogenic dendritic cells. Combined with high-content imaging analysis, this platform offers a powerful solution for preclinical translational research.}},
  author       = {{Agarkova, Irina and Rotankova, Nadezhda and Rudnik, Michal and Strebel, Silvan and Zimmermannova, Olga and Rosa, André and Fiúza Rosa, Fábio and Moritz, Wolfgang and Pereira, Carlos-Filipe and Pires, Cristiana}},
  issn         = {{1538-7445}},
  language     = {{eng}},
  month        = {{04}},
  note         = {{Conference Abstract}},
  number       = {{7 Supplement}},
  publisher    = {{American Association for Cancer Research Inc.}},
  series       = {{Cancer Research}},
  title        = {{Assessing efficacy and immune-stimulatory effects of tumor-derived dendritic cell reprogramming using immuno-competent 3D tumor spheroid model}},
  url          = {{http://dx.doi.org/10.1158/1538-7445.AM2023-4550}},
  doi          = {{10.1158/1538-7445.AM2023-4550}},
  volume       = {{83}},
  year         = {{2023}},
}