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

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.