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Assessing efficacy ...
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Agarkova, IrinaInSphero AG
(författare)
Assessing efficacy and immune-stimulatory effects of tumor-derived dendritic cell reprogramming using immuno-competent 3D tumor spheroid model
- Artikel/kapitelEngelska2023
Förlag, utgivningsår, omfång ...
Nummerbeteckningar
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LIBRIS-ID:oai:lup.lub.lu.se:50d2ed2c-5f58-48b7-9cef-fed2fabc2249
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https://lup.lub.lu.se/record/50d2ed2c-5f58-48b7-9cef-fed2fabc2249URI
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https://doi.org/10.1158/1538-7445.AM2023-4550DOI
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Språk:engelska
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Sammanfattning på:engelska
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Ämneskategori:ref swepub-contenttype
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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.
Ämnesord och genrebeteckningar
Biuppslag (personer, institutioner, konferenser, titlar ...)
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Rotankova, NadezhdaInSphero AG
(författare)
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Rudnik, MichalInSphero AG
(författare)
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Strebel, SilvanInSphero AG
(författare)
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Zimmermannova, OlgaLund University,Lunds universitet,Avdelningen för molekylärmedicin och genterapi,Institutionen för laboratoriemedicin,Medicinska fakulteten,Cellulär omprogrammering i hematopoes och immunitet,Forskargrupper vid Lunds universitet,LUCC: Lunds universitets cancercentrum,Övriga starka forskningsmiljöer,Division of Molecular Medicine and Gene Therapy,Department of Laboratory Medicine,Faculty of Medicine,Cell Reprogramming in Hematopoiesis and Immunity,Lund University Research Groups,LUCC: Lund University Cancer Centre,Other Strong Research Environments(Swepub:lu)ol1273zi
(författare)
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Rosa, AndréAsgard Therapeutics AB(Swepub:lu)an6072ro
(författare)
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Fiúza Rosa, FábioAsgard Therapeutics AB(Swepub:lu)fa8852fi
(författare)
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Moritz, WolfgangInSphero AG
(författare)
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Pereira, Carlos-FilipeLund University,Lunds universitet,Avdelningen för molekylärmedicin och genterapi,Institutionen för laboratoriemedicin,Medicinska fakulteten,Cellulär omprogrammering i hematopoes och immunitet,Forskargrupper vid Lunds universitet,WCMM- Wallenberg center för molekylär medicinsk forskning,LUCC: Lunds universitets cancercentrum,Övriga starka forskningsmiljöer,Division of Molecular Medicine and Gene Therapy,Department of Laboratory Medicine,Faculty of Medicine,Cell Reprogramming in Hematopoiesis and Immunity,Lund University Research Groups,WCMM-Wallenberg Centre for Molecular Medicine,LUCC: Lund University Cancer Centre,Other Strong Research Environments(Swepub:lu)ca4155pe
(författare)
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Pires, CristianaAsgard Therapeutics AB(Swepub:lu)cr5211fe
(författare)
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InSphero AGAvdelningen för molekylärmedicin och genterapi
(creator_code:org_t)
Sammanhörande titlar
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Ingår i:Cancer Research83:7 Supplement1538-7445
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Agarkova, Irina
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Rotankova, Nadez ...
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Rudnik, Michal
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Strebel, Silvan
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Zimmermannova, O ...
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Rosa, André
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visa fler...
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Fiúza Rosa, Fábi ...
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Moritz, Wolfgang
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Pereira, Carlos- ...
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Pires, Cristiana
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Cancer Research
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Lunds universitet