Name: Tractable In Vivo Reprogramming of Tumor Cells to Type 1 Conventional Dendritic Cell-like Cells (Video) | JoVE Journal
Uploaded: 2025-08-01T14:35:00.000+00:00
Duration: 10 min 4 s
Description: Learn in vivo reprogramming of tumor cells into cDC1-like dendritic cells using transcription factors PU.1, IRF8, and BATF3. This method converts immune cold tumors into immune hot, enhancing anti-tumor immunity.

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5 minutes

We thank Mariana Lopes and Camille Chatelain, members of the Cell Reprogramming in Hematopoiesis and Immunity Laboratory, for editing and proofreading the manuscript.
This study was accomplished within the context of the National ATMP Research School funded by the Swedish Research Council. The projects discussed here were co-funded by the European Research Council under the European Union&#39;s Horizon 2020 research and innovation program (866448-TrojanDC and 101189370), Novo Nordisk Fonden (NNF22C0079466), European Innovation Council (101130218-RESYNC), Cancerfonden (23 2932 Pj), the Swedish Research Council (2020-00615), Swedish Innovation Agency (2024-02178), ALF (44410), FCT (2022.02338.PTDC), and Plano de Recupera&#231;&#227;o e Resili&#234;ncia de Portugal pelo fundo NextGenerationEU (C644865576-00000005). The Knut and Alice Wallenberg Foundation, the Medical Faculty at Lund University, and Region Sk&#229;ne are acknowledged for generous financial support.
Materials Availablity:
Constructs and vectors used for reprogramming are available from Asgard Therapeutics under a material transfer agreement with the company. All other data needed to evaluate the conclusions in the paper are present in the main text or the supplementary materials.

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C.-F.P. has equity interest and serve in management positions at Asgard Therapeutics AB, which develops cancer immunotherapies based on in vivo DC reprogramming technologies. C.-F.P is an inventor on granted patents US 11,345,891, JP 7303743, and CN ZL201880005047.3 and patent applications WO 2018/185709 and WO 2022/243448 (together with E.A.) held by Asgard Therapeutics, which cover the cell-reprogramming approach described here.

In this protocol, we describe a tractable method to generate cDC1-like cells based on in vivo reprogramming of cancer cells from solid tumors. Our results demonstrate that the overexpression of PU.1, IRF8, and BATF3 drives the gradual acquisition of a cDC1 phenotype in the TME until day 9, whereafter cDC1-like cells are depleted from tumors. Ultimately, this leads to an increase in the infiltration of CD45 cells into the TME by day 3, causing the remodeling of an &#34;immune-cold&#34; TME to become &#34;immune-hot&#34;.
Current protocols to generate cDC1 include enrichment of autologous DCs from peripheral blood, differentiation from CD34+ hematopoietic progenitor cells, or differentiation from pluripotent stem cells14,15,16,17,18,19. However, these methods present limiting factors: insufficient maturation, reduced antigen presentation and cytokine/chemokine production capacity in vivo, susceptibility to immunosuppression, high DC heterogeneity, and low numbers of generated cells7. Here, we provide a protocol that allows the scalable generation of cDC1-like cells in vivo for functional and mechanistic studies.
With this method, we tested whether the expression of the transcription factor combination PU.1, IRF8, and BATF3 was sufficient to drive in vivo reprogramming. This protocol enables precise control over delivery efficiency, as varying the dose of genetically modified cells implanted directly influences treatment efficacy. We have previously shown that 2% transduced cells, which correspond to 0.15% CD45+ and MHC-II+ cDC1-like cells, are sufficient to elicit anti-tumor immunity20. Furthermore, the rapid immune infiltration observed on day 3 suggests that partially reprogrammed cDC1-like cells are functionally active in vivo, as there is no substantial increase in fully reprogrammed cDC1-like cells at this time point. In the future, it will be interesting to unravel the functional role of partially versus fully reprogrammed cDC1-like cells in anti-tumor efficacy using modified vectors containing a suicide gene to induce cell death in selected cell populations at defined time points24. This protocol can further be used to explore the combination with epigenetic adjuvants, which can increase reprogramming efficiency25. Finally, this method could also be adapted to generate other DC subtypes in vivo, launching different types of immune responses, for example, anti-viral or tolerogenic responses14. In addition, we can harness in vivo reprogramming as a platform to perform barcoded screens for transcription factor combinations to generate diverse immune cell types. This possibility could increase the flexibility of treatments by leveraging diverse functional properties of specific immune subsets.
In our previous study, we demonstrated that the immunosuppressive in vivo environment does not hinder the reprogramming of human cancer cells into immunogenic cDC1s20. In this article, we show that in mice, reprogramming is more rapid and efficient in vivo than in vitro, suggesting that the enhanced reprogramming observed in vivo is a conserved phenomenon across both species. However, one of the limitations of this method is the lack of the in vivo environment during initial vector delivery; thus, it cannot discern whether environmental cues impact in vivo delivery efficiency. This should be determined in situ by comparing different moieties. Adenoviral vectors, which we have identified to be more efficient than lentiviral vectors for in situ delivery20&#160;could be compared to non-viral moieties such as linear, circular, and self-amplifying RNAs or small molecule cocktails26,27.
We have previously shown that cDC1 reprogramming endows cancer cells with pro-inflammatory cytokine and lymphocyte-recruiting chemokine secretion and professional antigen processing and presentation on MHC-I and MHC-II in melanoma models23,28. This results in remodeling of the TME from an &#34;immune-cold&#34; to an &#34;immune-hot&#34; state by inducing TLS formation, which increases the infiltration of cytotoxic effector T cells, NK cells, and B cells, and reduces immunosuppressive populations such as regulatory and exhausted T cells20. In patients, positive responses to immunotherapy have been linked to the presence of TLS within tumors, where sustained T and B cell priming against tumor antigens generates tumor-specific antibody responses29,30. In the future, we envision employing this tractable in vivo cDC1 reprogramming protocol to unravel the mechanisms of induced TLS neogenesis, TME remodeling and anti-tumor immunity across different cancer types and characterize whether the production of tumor-specific antibodies is stimulated.
We have previously observed synergism between in vivo cDC1 reprogramming and ICB with anti-PD-1 or anti-CTLA-420. This suggests that the &#34;immune-hot&#34; TME created by cDC1 reprogramming could provide a favorable environment for possible combinations with other immunotherapies such as ACT including T cell receptor (TCR)-T cells, chimeric antigen receptor (CAR)-T cells, or tumor-infiltrating lymphocytes (TILs). Another advantage of this method is the possibility to perform large unbiased combinatorial or CRISPR screens, in which immunological receptors, cytokines, chemokines, or lymphotoxins could be overexpressed or depleted in cDC1-like cells in resistant tumor types, thus unraveling the role of distinct pathways in enhancing cDC1-mediated immunity. In conclusion, the current protocol provides a valuable resource for a broad scientific community, since in vivo cDC1 reprogramming is a flexible and powerful platform for mechanistic studies and therapeutic innovation in cancer immunotherapy.

Recent advancements in cancer immunotherapy modalities, including immune checkpoint blockade (ICB), adoptive cell therapy (ACT), and vaccines, have shifted the landscape of cancer treatment1. In melanoma, response rates to programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) inhibition can reach up to 60%2. Nevertheless, over 40% of melanoma patients fail to respond to ICB, and response rates in less immunogenic cancers, such as breast cancer, microsatellite-stable colorectal cancer, and glioblastoma, remain below 5%, highlighting clinical gaps3,4,5. A major barrier to broad clinical success is the poor activation of cytotoxic T cell responses, which depend on efficient antigen presentation within the tumor microenvironment (TME)6.
Conventional type 1 dendritic cells (cDC1s) are rare, but indispensable for the initiation of tumor-specific immunity, offering three key signals to prime T cell responses: (1) professional processing and presentation of antigens on major histocompatibility complexes (MHC) class I and II, (2) co-stimulation via CD80 and CD86, and (3) pro-inflammatory cytokine signaling including IL-127,8. Among the dendritic cell (DC) subsets, cDC1s excel in cross-presenting tumor cell-associated antigens to prime na&#239;ve or re-activate memory CD8+ T cells or drive CD4+ T cell differentiation to type 1 T helper (TH1) cells9. Additionally, cDC1s are the main producers of chemokines, C-X-C motif chemokine ligands 9 and 10 (CXCL9/10), which mediate T cell recruitment to the tumor tissue10,11,12,13. Over the last few years, numerous studies have highlighted that tumor rejection and effective responses to ICB or ACT strongly correlate with the presence of intratumoral cDC1s12,13. Hence, cDC1s have developed as an attractive target for cancer immunotherapy and successfully harnessing their unique features can have significant translational potential.
Current methods to generate cDC1s are based on blood isolation or differentiation from CD34+ progenitors or pluripotent stem cells14,15,16. The predominant clinical strategies rely on isolating a mixture of autologous DC subsets from the blood and stimulating them ex vivo with toll-like receptor agonists or pro-inflammatory cytokines to induce a mature, immunogenic program before reinfusion16. However, these strategies are limited by poor cell maturation, immunosuppressive barriers in the TME, and subset heterogeneity in the infused DC mixture, where only a small proportion of cDC1s is present7. Moreover, isolating only cDC1s for cancer immunotherapy or vaccination is challenging due their extremely low abundance in peripheral blood (&#60;0.3%)16. Additionally, cDC1s can be produced from CD34+ progenitor cells or induced pluripotent stem cells using cytokines and NOTCH signaling. However, these methods require complex and long protocols and yield low numbers and heterogeneous populations of DC subsets17,18,19.
To address these limitations, in vivo reprogramming provides a suitable strategy for cancer immunotherapy to generate cDC1-like cells, which resemble the phenotypic, transcriptional, and functional properties of cDC1s20. Through the overexpression of the cDC1-specific minimal gene regulatory network -- PU.1, IRF8, and BATF3 (collectively referred to as PIB) -- fibroblasts, stromal cells, and cancer cells have been gradually reprogrammed into cDC1-like cells enabling tumor antigen processing and presentation on MHC class I and II, co-stimulatory signaling via CD80 and CD86, and cytokine/chemokine secretion, including CXCL9/10 and IL-1220,21,22,23. cDC1 reprogramming progressed faster and more efficiently across human xenografts in vivo and independently of immunosuppression, generating cDC1-like cells with a mature immunogenic signature20. In syngeneic melanoma tumors, we observed that cDC1-like cells remodeled their TME, induced the formation of tertiary lymphoid structure (TLS), and recruited and expanded polyclonal TH1 and cytotoxic memory T cells, leading to long-term systemic immunity20. Finally, we developed a novel cancer immunotherapy modality based on in situ reprogramming of cancer cells into cDC1-like cells by intratumoral injection of adenoviral vectors20. These findings pave the way for clinical translation of cDC1 reprogramming as a new cancer immunotherapy modality that can harness the functional properties of cDC1s.
Here, we present a tractable protocol to generate cDC1-like cells within the TME based on in vivo cDC1 reprogramming. Lentiviral delivery of PIB to cancer cells and subsequent implantation as a defined mix of transduced cancer cells with untransduced parental cancer cells allows in vivo reprogramming of a controlled fraction of cancer cells within the TME. Additionally, this method is not limited by the in situ delivery efficiency of viral vectors20. We outline the steps for efficient lentiviral vector production, functional titration, and in vivo cDC1 reprogramming using the syngeneic melanoma model, YUMM1.7, as an example. We detail the experimental setup and the gating strategy for analyzing in vivo reprogrammed cDC1-like cells by flow cytometry after tumor dissociation. We also provide the steps to evaluate changes in the TME by performing cryopreservation, sectioning, and immunofluorescence staining of tumor sections, followed by confocal microscopy. Finally, we address how to monitor cDC1-induced immunity by measuring tumor growth and survival. This tractable and scalable approach offers researchers a valuable tool to assess combinatorial treatment strategies with in vivo cDC1 reprogramming, to characterize changes in the TME and TLS neogenesis across cancer models or perform large screens modifying the expression of various immune pathways, for example, antigen presentation or cytokine/chemokine secretion, and unravel their impact on antitumor immunity.

<table><tbody><tr><td>0.45 &amp;mu;m low-protein binding filter, 150 mL Bottle Top Vacuum Filter</td><td>Fisher scientific</td><td>15983297</td><td></td></tr><tr><td>2-Mercaptoethanol 50 mM</td><td>ThermoFisher Scientific</td><td>31350010</td><td></td></tr><tr><td>Anti-mouse CD45 Monoclonal Antibody (clone: 30-F11), Rat IgG2b, &amp;kappa;</td><td>ThermoFisher Scientific</td><td>14-0451-82</td><td></td></tr><tr><td>B6.SJL-PtprcaPepcb/BoyCrl (CD45.1)</td><td>The Jackson Laboratory</td><td>002014</td><td></td></tr><tr><td>BD Micro-Fine U-100 Insulin Syringes (0.5 mL)</td><td>Fisher Scientific</td><td>11395751</td><td></td></tr><tr><td>C57BL/6J mice</td><td>The Jackson Laboratory</td><td>000664</td><td></td></tr><tr><td>Collagenase D</td><td>Sigma Aldrich</td><td>11088882001</td><td></td></tr><tr><td>Corning Falcon Cell Strainer For Use With 50ml Conical Tubes</td><td>Sigma Aldrich</td><td>CLS352340</td><td></td></tr><tr><td>Corning cover glasses 24 x 50 mm&amp;nbsp;</td><td>Sigma Aldrich</td><td>CLS2975245</td><td></td></tr><tr><td>DAPI (4&#039;,6-diamidino-2-phenylindole, dihydrochloride)</td><td>ThermoFisher Scientific</td><td>D1306</td><td></td></tr><tr><td>DNase I</td><td>Sigma Aldrich</td><td>10104159001</td><td></td></tr><tr><td>eBioscience Fixable Viability Dye eFluor 450</td><td>Invitrogen</td><td>65-0863-14</td><td></td></tr><tr><td>eBioscience IHC Antigen Retrieval Solution - Low pH (10x)</td><td>ThermoFisher Scientific</td><td>00-4955-58</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>ThermoFisher Scientific</td><td>A5256701</td><td></td></tr><tr><td>Fluoromount-G Mounting Medium</td><td>ThermoFisher Scientific</td><td>00-4958-02</td><td></td></tr><tr><td>GFP Antibody [Alexa Fluor 594]</td><td>Novus Biologicals</td><td>NB600-308AF594</td><td></td></tr><tr><td>Gibco DMEM/Nutrient Mixture F-12 (DMEM/F-12)</td><td>ThermoFisher Scientific</td><td>31331028</td><td></td></tr><tr><td>Gibco MEM Non-Essential Amino Acids Solution (100x)</td><td>ThermoFisher Scientific</td><td>11140035</td><td></td></tr><tr><td>Gibco Opti-MEM I</td><td>ThermoFisher Scientific</td><td>11058021</td><td></td></tr><tr><td>Gibco Roswell Park Memorial Institute (RPMI) 1640 Medium</td><td>ThermoFisher Scientific</td><td>52400025</td><td></td></tr><tr><td>Gibco Sodium Pyruvate (100 mM)</td><td>ThermoFisher Scientific</td><td>11360070</td><td></td></tr><tr><td>Gibco Trypan Blue Solution, 0.4%</td><td>ThermoFisher Scientific</td><td>15250061</td><td></td></tr><tr><td>GlutaMAX Supplement&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>35050061</td><td></td></tr><tr><td>Goat anti-rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Polyclonal, Alexa Fluor 488</td><td>ThermoFisher Scientific</td><td>A-11006</td><td></td></tr><tr><td>HEK 293T cells</td><td>ATTC</td><td>CRL-11268</td><td></td></tr><tr><td>Hexadimethrine bromide (Polybrene)</td><td>Sigma Aldrich</td><td>H9268</td><td></td></tr><tr><td>HyClone Dulbecco&#039;s Modified Eagle Medium (DMEM) with high glucose</td><td>Cytiva</td><td>SH30243.01</td><td></td></tr><tr><td>HyClone Penicillin Streptomycin 100x Solution (Pen/Strep)</td><td>Cytiva</td><td>SV30010</td><td></td></tr><tr><td>HyClone Phosphate Buffered Saline solution (PBS)</td><td>Cytiva</td><td>SH30028.02</td><td></td></tr><tr><td>Ketaminol vet. Solution for injection 100 mg/mL</td><td>MSD Animal Health Sweden</td><td>QN01AX03</td><td></td></tr><tr><td>Microtome Blade - C35</td><td>pfm medical</td><td>207500005</td><td></td></tr><tr><td>Mouse anti-CD45.2 (104), Surface, APC, Monoclonal</td><td>Biolegend</td><td>109814</td><td></td></tr><tr><td>Mouse anti-MHCII (M5/114.15.2), Surface, PE-Cy7, Monoclonal</td><td>Biolegend</td><td>107630</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Abcam</td><td>ab7481</td><td></td></tr><tr><td>Normal Rat Serum (sterile)</td><td>Abcam</td><td>ab7488</td><td></td></tr><tr><td>Open-Top Thinwall Polypropylene Tubes</td><td>Beckman Coulter Life Sciences</td><td>326823</td><td></td></tr><tr><td>Paraformaldehyde, 16% w/v aq. soln., methanol free</td><td>ThermoFisher Scientific</td><td>043368.9M</td><td></td></tr><tr><td>pFUW-tetO-Batf3, a monocistronic cassette encoding mouse Batf3</td><td>Addgene</td><td>139837</td><td></td></tr><tr><td>pFUW-tetO-Irf8, a monocistronic cassette encoding mouse Irf8</td><td>Addgene</td><td>139838</td><td></td></tr><tr><td>pFUW-tetO-Pu.1, a monocistronic cassette encoding mouse Pu.1</td><td>Addgene</td><td>139839</td><td></td></tr><tr><td>Plasmid pMD2.G</td><td>Addgene</td><td>12259</td><td></td></tr><tr><td>Plasmid psPAX2</td><td>Addgene</td><td>12260</td><td></td></tr><tr><td>Plasmid SFFV-eGFP (eGFP)</td><td>In house cloning</td><td>-</td><td></td></tr><tr><td>Plasmid SFFV-mCherry (mCherry)</td><td>In house cloning</td><td>-</td><td></td></tr><tr><td>Plasmid SFFV-PIB-eGFP,&amp;nbsp; a polycistronic cassette encoding mouse PU.1, IRF8, and BATF3 (PIB)&amp;nbsp;</td><td>In house cloning</td><td>-</td><td>Constructs and vectors used for reprogramming are available from Asgard Therapeutics under a material transfer agreement with the company</td></tr><tr><td>Polyethylenimine (PEI), Linear, MW 25000, Transfection Grade</td><td>Polysciences</td><td>23966</td><td></td></tr><tr><td>ReadyProbes Hydrophobic Barrier Pap Pen</td><td>ThermoFisher Scientific</td><td>R3777</td><td></td></tr><tr><td>Sodium Butyrate</td><td>Sigma Aldrich</td><td>303410</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S7903</td><td></td></tr><tr><td>SuperFrost Plus Adhesion Microscope Slides</td><td>Epredia</td><td>J1800AMNZ</td><td></td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>Sakura Finetek</td><td>4583</td><td></td></tr><tr><td>TrypLE Express Enzyme (1x) no phenol red</td><td>ThermoFisher Scientific</td><td>12604021</td><td></td></tr><tr><td>TT Cryomold Intermediate, square (15 x 15 x 5 mm)</td><td>Sakura Finetek</td><td>4566</td><td></td></tr><tr><td>UltraPure 0.5 M EDTA, pH 8.0</td><td>Invitrogen</td><td>15575020</td><td></td></tr><tr><td>Xysol vet. Solution for injection 20 mg/mL</td><td>VM Pharma</td><td>QN05CM92</td><td></td></tr><tr><td>YUMM1.7 cells</td><td>Antineo</td><td></td><td></td></tr></tbody></table>

tractable in vivo reprogramming of tumor cells to type 1 conventional dendritic cell-like cells

Tractable In Vivo Reprogramming of Tumor Cells to Type 1 Conventional Dendritic Cell-like Cells

The efficacy of cancer immunotherapy relies on the recruitment and activation of cytotoxic T cell responses against solid tumors by type 1 conventional dendritic cells (cDC1s). However, the generation of cDC1s for cancer immunotherapy faces significant limitations, including poor cell yield, functional heterogeneity, and susceptibility to immunosuppression in the tumor microenvironment (TME). We recently developed an immunotherapy modality based on in vivo reprogramming of cancer cells into immunogenic cDC1-like cells, which enabled cancer cells to present tumor antigens as cDC1s and elicited polyclonal cytotoxic T cell responses and durable systemic anti-tumor immunity. Here, we describe a tractable protocol to generate cDC1-like cells within the TME by overexpressing the minimal cDC1-specific gene regulatory network-PU.1, IRF8, and BATF3 (collectively referred to as PIB)-in cancer cells, followed by subcutaneous implantation of a mixture of transduced and parental cells. PIB overexpression drives the gradual acquisition of the hematopoietic marker CD45 and the professional antigen presentation complex MHC class II on tumor cells, serving as cell surface readouts for in vivo cDC1 reprogramming. When compared to the reprogramming process in vitro, reprogramming of the YUMM1.7 mouse melanoma model in vivo demonstrated faster kinetics and higher efficiency. cDC1-like cells induced rapid remodeling of the TME by recruiting host immune cells within the first 3 days and leading to the formation of tertiary lymphoid structure by day 9. Reprogrammed cDC1-like cells persisted in tumors for at least 9 days but were undetected at day 15. The in vivo cDC1 reprogramming protocol described here provides a tractable and robust method to effectively transform "immune-cold" tumors into "immune-hot". Overall, it offers a powerful platform to study the mechanisms underlying cDC1-mediated anti-tumor immunity and uncover synergistic combinations with other cancer immunotherapy modalities.

A schematic overview of the tractable in vivo reprogramming protocol is presented in Figure 1. First, we produced the polycistronic lentiviral vector SFFV-PIB-IRES-eGFP (PIB-eGFP), encoding for the cDC1-instructing reprogramming factors PU.1, IRF8, BATF3, followed by an internal ribosomal entry site (IRES) and enhanced green fluorescent protein (eGFP) in the HEK 293T packaging cell line. To control for lentivirus-mediated immunogenicity, we produced an empty SFFV-MCS-eGFP (eGFP) vector, containing a multiple cloning site (MCS) and IRES-eGFP, but excluding the reprogramming factors. Following viral production, we harvested the lentivirus-containing supernatant and concentrated it by ultracentrifugation. We functionally titrated the virus to determine the amount required to achieve a 1:1 ratio of transduced (eGFP+) to untransduced (eGFP-) cells, which we evaluated on day 3 post transduction. Then, we used the resulting virus amount to transduce the selected cancer cell line on day -1. After 24 h, we collected the cells and implanted them subcutaneously to establish tumors. This strategy ensured that transduced cells reprogrammed into cDC1-like cells in vivo, within the TME. At indicated time points post engraftment, we excised and processed tumors for downstream analyses, including flow cytometry of dissociated tumor cells, to quantify in vivo reprogramming efficiency, or immunofluorescence staining of cryopreserved tumor sections to characterize changes in the TME.
First, to ensure the production of high lentiviral titers, we monitored HEK 293T cells before transfection, targeting to reach 70-80% confluency and 24 h later, we assessed the transfection efficiency through eGFP expression by fluorescence microscopy (Figure 2A). Next, we collected virus-containing medium 48 h and 60 h post transfection, ultracentrifuged it, and stored in aliquots at -80 °C (Figure 2B). Then, we determined the optimal viral amount required to achieve a 1:1 ratio of eGFP+ to eGFP- cells in YUMM1.7 melanoma cells. We seeded 105 YUMM1.7 cells per well in a 6-well plate, transduced the cells with increasing volumes of lentivirus, and replaced the virus-containing supernatant with fresh DMEM/F12 complete media after 24 h (Figure 2C). On day 3, we assessed eGFP expression by fluorescence microscopy, before proceeding to quantify the precise percentages of eGFP+ cells by flow cytometry (Figure 2C). To quantify the ratio of eGFP+ to eGFP- cells, we gated in live, single cells to exclude dead cells and doublets (Figure 2D). The optimal volume of the produced lentivirus batch was determined both for PIB-eGFP and the control eGFP virus as 2 µL per 105 YUMM1.7 cells, resulting in 50.0 ± 0.3% and 63.0 ± 0.9% eGFP+ cells, respectively (Figure 2E).
Next, to evaluate in vivo reprogramming efficiency, we generated a fluorescently labelled (mCherry+) cancer cell line (YUMM1.7-mCherry). This ensured that we could discriminate between cancer cells and stromal or immune cells by gating in live mCherry+ cells in the flow cytometry analysis (Figure 3A,B). We then transduced YUMM1.7-mCherry cells with 2 µL of PIB-eGFP or control eGFP virus per 105 cells to achieve a 1:1 eGFP+ to eGFP- ratio. We subcutaneously implanted 1.6 × 106 cells per tumor in CD45.1 mice and kept an aliquot of cells to culture in vitro for comparison. We evaluated reprogramming efficiency by flow cytometry, gating live mCherry+ eGFP+ and CD45.1- cells to exclude host immune cells that have engulfed eGFP or mCherry (Figure 3B). From days 1 to 9, we observed the gradual acquisition of CD45.2 and/or MHC-II expression, marking partially reprogrammed cDC1-like cells (CD45.2+ MHC-II- or CD45.2- MHC-II+) and fully reprogrammed cDC1-like cells (CD45+ MHC-II+) (Figure 3C). We validated that reprogramming of mouse cancer cells in vivo resulted in higher percentages of cDC1-like cells at day 9 compared to in vitro conditions (9.54 ± 1.54% CD45+ MHC-II+ in vivo vs. 4.72 ± 1.1 % CD45+ MHC-II+in vitro, P = 0.018). The kinetics of reprogramming were also accelerated as we observed a higher percentage of cDC1-like cells as early as day 4 (8.7 ± 3.3% CD45+ MHC-II+in vivo vs. 0.3 ± 0.1 % CD45+ MHC-II+in vitro, P = 0.05), indicating that the presence of environmental cues promote faster and more efficient reprogramming towards cDC1-like cells19. While in vitro reprogrammed cDC1-like cells were present until day 15, in vivo cDC1-like cells persisted for 9 days, but were undetected by day 15, suggesting depletion from the TME by the host immune system (Figure 3C).
Lastly, we aimed to characterize early changes of the TME as a result of in vivo cDC1 reprogramming in the first 9 days. We performed immunofluorescence staining of cryopreserved and sectioned tumor tissues from both eGFP and PIB-eGFP tumors after 3 and 9 days of in vivo reprogramming, focusing on the infiltration of immune cells marked by the pan-hematopoietic marker CD45. Notably, cDC1-like cells recruited significantly more CD45 cells to the TME readily on day 3, compared to eGFP tumors (49.6 ± 17.0 vs. 8.0 ± 5.8 eGFP mean fluorescence intensity of CD45 staining per tumor area; Figure 4A,B). Interestingly, we detected TLS within the TME of tumors after 9 days of in vivo reprogramming. This suggests that cDC1-like cells can rapidly remodel the TME from an "immune-cold" to an "immune-hot" environment within 3 days, which then subsequently leads to TLS formation on day 9.
<img alt="Polycystronic lentiviral vector experiment diagram; cell transduction, reprogramming, tumor analysis." class="xfigimg" src="/files/ftp_upload/68739/68739fig1.jpg" data-altupdatedat="1762509930000" data-alt="Figure 1"> 
Figure 1: Protocol overview to generate cDC1-like cells by in vivo reprogramming of cancer cells. Lentiviral vectors SFFV-PIB-IRES-eGFP-encoding PU.1, IRF8, and BATF3 in a polycistronic cassette under the control of the spleen focus-forming virus promoter (SFFV) and eGFP after an internal ribosome entry site (IRES) to track transduced cells by flow cytometry and fluorescence microscopy-are produced in the HEK 293T packaging cell line. Then, lentiviral vectors are ultracentrifuged and functionally titrated to determine the amount of virus required for transducing cancer cells in a ratio of 1:1 transduced eGFP+ to untransduced eGFP- cells. For in vivo reprogramming experiments, cancer cells are plated with the optimal amount of lentivirus on day -1. The next day, this 1:1 mix of transduced GFP+ and untransduced GFP- cells is washed and collected for subcutaneous tumor establishment in mice. Parallel in vitro cultures are maintained to validate transduction on day 3 by flow cytometry. Animals are monitored every 2-3 days for tumor growth and survival. Tumors are collected at defined time points, dissociated and stained for surface marker expression for flow cytometry analysis. Alternatively, whole tissues are fixed, cryopreserved, and sectioned for immunofluorescence staining and confocal microscopy analysis. In vivo reprogramming efficiency is evaluated by flow cytometry analysis, while changes in the tumor microenvironment are characterized by confocal imaging. <a href="https://www.jove.com/files/ftp_upload/68739/68739fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Lentivirus production in HEK 293T cells, chart showing transfection and ultracentrifuge steps." class="xfigimg" src="/files/ftp_upload/68739/68739fig2.jpg" data-altupdatedat="1762509930000" data-alt="Figure 2"> 
Figure 2: Production and functional titration of lentivirus for in vivo cDC1 reprogramming. (A) When HEK 293T cells reach ~70-80% confluency, they are transfected with a mixture containing 10 µg of transfer plasmid either SFFV-PIB-eGFP (PIB-eGFP) or empty lentiviral vector control SFFV-eGFP (eGFP), along with 7.5 µg of psPAX2, and 2.5 µg of mPD2.G and 60 µL of 1 mg/mL PEI. Brightfield and fluorescence microscopy images of HEK 293T cells showing eGFP expression 24 h post transfection, indicating successful plasmid delivery. Scale bars = 200 µm. (B) Schematic representation of the ultracentrifugation workflow to concentrate lentivirus. The lentivirus-containing supernatant of HEK 293T cells is collected 48 h and 60 h post transfection, filtered through 0.45 µm low-protein binding PES filter, and 37 g are weighed in Open-Top Thinwall Polypropylene Tubes using a bench scale. Tubes are ultracentrifuged in a Swinging-Bucket Rotor, the supernatant is aspirated, and the pellet dried for 5 min. Pellets are then resuspended in ice-cold DMEM containing HEPES and incubated overnight before aliquoting and storing at -80 °C. (C) Experimental strategy of the functional titration to determine the optimal viral dose for achieving a 1:1 ratio of GFP+ to GFP- cells. Brightfield and fluorescence microscopy images showing eGFP expression in YUMM1.7 melanoma cells, 72 h after transduction, indicating successful delivery of the reprogramming factors and eGFP. Scale bars = 100 µm. (D) Representative flow cytometry plots showing gating strategy to quantify the ratio of eGFP+ cells. Live cells were gated for viability dye negative population (DAPI) followed by doublet exclusion. (E) Flow cytometry quantification of transduction efficiency with increasing viral doses to determine the optimal volume for achieving 1:1 GFP+/GFP- cells. For both PIB-eGFP and eGFP lentivirus, 2 µL volume per 105 cells achieved the desired ratio. Abbreviations: cDC1s = type 1 conventional dendritic cells; DAPI = 4',6-diamidino-2-phenylindole; PIB = PU.1, IRF8, and BATF3; PEI = polyethylenimine. <a href="https://www.jove.com/files/ftp_upload/68739/68739fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="In vivo cDC1 reprogramming, flow cytometry analysis; involves PIB-eGFP, CD45.1 mice; charts." class="xfigimg" src="/files/ftp_upload/68739/68739fig3.jpg" data-altupdatedat="1762509930000" data-alt="Figure 3"> 
Figure 3: Assessing in vivo cDC1 reprogramming efficiency. (A) Schematic representation of the experimental workflow for evaluating in vivo cDC1 reprogramming efficiency. Cancer cells are initially transduced with a lentivirus expressing mCherry to generate a fluorescently labeled cell line (YUMM1.7-mCherry), followed by transduction with the optimized dose of PIB-eGFP lentivirus on day -1. Cells are then injected subcutaneously with a 1:1 ratio of eGFP+/eGFP- cells on day 0 to establish tumors in CD45.1 mice and allow PIB-transduced cells to reprogram into cDC1-like cells within the TME. Tumors are then isolated at indicated time points, dissociated, and stained for CD45.1, CD45.2, and MHC-II using flow cytometry. This allows for the exclusion of CD45.1 host immune cells to quantify in vivo cDC1 reprogramming efficiency by expression of CD45.2 and MHC-II. Parallel in vitro cultures are kept throughout the time points as a comparison to in vivo reprogramming. (B) Representative flow cytometry plots illustrating the gating strategy on day 9. Cells were gated on mCherry+ and GFP+ to identify successfully transduced cancer cells. Host immune cells were excluded by gating out CD45.1+ cells. (C) Quantification of in vivo reprogramming efficiency (left) of YUMM1.7-mCherry cells by flow cytometry as the percentage of CD45+ MHC-II+ cells (completely reprogrammed) and CD45.2+ HLA-DR- or CD45.2- HLA-DR+ cDC1-like cells (partially reprogrammed) gated within eGFP+ mCherry+ cells. Representative flow cytometry plots (right) showing the gradual acquisition of CD45.2 and MHC-II expression in PIB-eGFP-transduced YUMM1.7-mCherry cells throughout the indicated time points. eGFP-transduced YUMM1.7-mCherry cells were used as a control and in vitro reprogrammed cDC1-like cells were used for comparison. Data in (C) indicate mean ± SD of 3-5 biological replicate experiments. Statistical analysis in (C) was performed for CD45+ MHC-II+ cells using the Mann-Whitney test. *P &lt; 0.05. Abbreviations: cDC1s = type 1 conventional dendritic cells; PIB = PU.1, IRF8, and BATF3; TME = tumor microenvironment. <a href="https://www.jove.com/files/ftp_upload/68739/68739fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Tumor microenvironment remodeling: fluorescence microscopy of eGFP, CD45; analysis chart included." class="xfigimg" src="/files/ftp_upload/68739/68739fig4.jpg" data-altupdatedat="1762509930000" data-alt="Figure 4"> 
Figure 4: Analyzing immune infiltration in the tumor microenvironment following in vivo cDC1 reprogramming. (A) Representative confocal images showing CD45+ cells in YUMM1.7 tumors after subcutaneous implantation of PIB-eGFP- or control eGFP-transduced cells (1:1 ratio of transduced to parental cells) on days 3 and 9. Scale bars = 500 µm. (B) Quantification of the MFI for CD45 staining in PIB-treated and control eGFP-transduced YUMM1.7 tumors on days 3 and 9. Data in (B) indicate mean ± SD of 3-6 biological replicate experiments. Statistical analysis in (B) was performed using the Mann-Whitney test. *P &lt; 0.05 and ***P &lt; 0.001. Abbreviations: cDC1s = type 1 conventional dendritic cells; PIB = PU.1, IRF8, and BATF3; MFI = mean fluorescence intensity. <a href="https://www.jove.com/files/ftp_upload/68739/68739fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

This protocol describes the in vivo reprogramming of mouse cancer cells into type 1 dendritic-like cells within the tumor microenvironment through enforced expression of the transcription factors PU.1, IRF8, and BATF3.

The efficacy of cancer immunotherapy relies on the recruitment and activation of cytotoxic T cell responses against solid tumors by type 1 conventional ...

We investigate in vivo programming of cancer cells into cDC1-like cells as a novel immunotherapy strategy. We aim to determine whether this approach can convert immune cold tumors into immune hot, thereby announcing anti-tumor immunity. We address limitations of current in vivo cDC1 generation strategies by presenting a protocol to convert cancer cells into immunogenic cDC1-like cells directly within the tumor, enabling the study of cDC1-driven immune responses in situ.Our tractable in vivo programming protocol reliably generates mature immunogenic cDC1-like cells, overcoming the suppressive tumor microenvironment, and because these cells are generated directly in vivo, this approach eliminates the need for in vitro immune cell manufacturing. Our findings establish a foundation for translating in vivo transcription factor-mediated reprogramming to the clinic, introducing off-the-shelf, yet personalized cancer immunotherapy that elicits durable anti-tumor immune responses. Moving forward, we're investigating how cDC1-like cells reprogramming can elicit the formation of tertiary and infrastructure and whether this translates into humoral immunity.Additionally, we're also exploring its potential synergy with other immunotherapeutic approaches. To begin, culture the HEK 293T cells until they reach 70 to 80%confluency. Now, vortex the prepared transfection mix containing the transfer plasmid thoroughly for one minute and incubate the mixture for 15 minutes at room temperature to allow DNA lipid complex formation.Aspirate the medium from the HEK 293T cells and add 10 milliliters of DMEM without penicillin streptomycin and supplemented with 10%FBS. Then, add the transfection mix dropwise to the 150 millimeter plates and incubate for 16 hours at 37 degrees Celsius with 5%carbon dioxide. After incubation, replace the medium with 20 milliliters of DMEM complete medium supplemented with one millimolar sodium butyrate, and incubate the cells for 24 hours at 37 degrees Celsius with 5%carbon dioxide.Next, collect the lentivirus-containing supernatant into a 50 milliliter conical tube at 48 and 60 hours post-transfection. After the first collection, add 12 milliliters of prewarmed complete DMEM per 150 millimeter plate for the second harvest. Filter the collected liquid through a 0.45 micrometer, low protein binding polyethersulfone filter.Pipette 37 grams of the lentivirus-containing medium into 38.5 milliliter open top thin wall polypropylene tubes. Centrifuge the tubes in a swinging bucket rotor at 25, 000 G for 90 minutes at four degrees Celsius. After centrifugation, aspirate the supernatant without disturbing the pellet.Place the open top thin wall polypropylene tubes upside down on a laboratory wipe or similar absorbent material for five minutes to drain excess liquid and dry the pellet. Next, add 200 microliters of ice cold DMEM supplemented with HEPES or PBS to each pellet and transfer the mixture to a microcentrifuge tube placed in a 50 milliliter conical tube. Incubate the pellet overnight at four degrees Celsius to allow complete resuspension.Combine all the resuspended pellets into a single container, aliquot the viral stock into smaller volumes between 50 and 200 microliters, and store at minus 80 degrees Celsius for long-term use. After cancer cell expansion, prepare the cell suspensions for transduction to achieve a near one-to-one ratio of EGFP positive to EGFP negative cells on day three. Add eight micrograms per milliliter of polybrene to the cell suspension, followed by the appropriate volume of lentivirus, as previously determined.Mix the contents gently without generating bubbles. Now, pipette two milliliters of the prepared cell suspension containing one times 10 to the power of five cells into each well of a six-well tissue culture plate and incubate for 24 hours at 37 degrees Celsius with 5%carbon dioxide. After 24 hours, remove the lentivirus-containing medium from each well.Wash the wells two times with PBS. Add 500 microliters of trypsin to each well and incubate for five to 10 minutes at 37 degrees Celsius with 5%carbon dioxide. Next, examine the wells under a light microscope to confirm complete cell detachment from the plate.Then, collect the cells using complete media into 50 milliliter conical tubes. Centrifuge the cells for five minutes at 350 G to pellet them. Wash the pellet with PBS.Take an aliquot and assess viable cell count using a hemocytometer and trypan blue staining. Next, resuspend the final cell pellet in ice cold PBS to achieve a concentration of two to four times 10 to the power of seven cells per milliliter, corresponding to one to two times 10 to the power of six cells in 50 microliters per tumor. Place the cells on ice and relocate to the animal experiment area.After anesthetizing the CD45.1 mouse, place it on a warming pad to prevent hypothermia. Apply eye cream to the mouse to prevent ocular dehydration during anesthesia. Using a razor, shave the right flank of the mouse.Optionally, mark the mouse with an ear clipper. Disinfect the shaved area and remove remaining hair with a 70%ethanol dipped swab. Now, using a P1000 pipette, resuspend the cancer cells thoroughly.Load the fully resuspended cell suspension into a 0.5 milliliter syringe fitted with a 30 gauge needle. Gently lift the skin on the shaved flank of the mouse using two fingers and insert the syringe needle tip into the crease formed by the lifted skin. Release the lifted skin while holding the syringe in place with one hand.Use the other hand to stretch the skin top with two fingers. Slowly inject 50 microliters of the cancer cell suspension to create a visible oval-shaped subcutaneous swelling at the injection site. After the injection procedure, monitor the animal until it fully awakens from anesthesia to ensure survival.HEK 293T cells transfected with SFFV-PIB-eGFP plasmid showed clear eGFP expression 24 hours post-transfection, confirming successful plasma delivery. 72 hours after transduction, melanoma cells indicated successful delivery of the reprogramming factors and eGFP. On day nine post-implantation of cells, in vivo reprogramming yielded a higher percentage of fully reprogrammed CD45-positive, MHC-II-positive, cDC1-like cells than in vitro conditions.On day three after the implantation, PIB-eGFP tumors showed markedly higher CD45-positive immune cell infiltration than eGFP controls, indicating early immune remodeling. By day nine, CD45 mean fluorescence intensity per tumor area remained significantly higher in PIB-eGFP tumors than in controls. Tertiary lymphoid structures became detectable in PIB-eGFP tumors by day nine, indicating structural immune changes in the tumor microenvironment.

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Cancer Research

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