GTGG-3' (Hs.Cas9.OGDH.1.AB) 5'-ACGAATGCCGGAGCCCCACCGG-3'
(Hs.Cas9.OGDH.1.AC).

qRT-PCR for mitochondrial DNA

DNA extraction was performed with FastPure Blood/Cell/Tissue/Bacteria DNA Isolation Mini Kit (Vazyme Biotech DC112-01). Cells were lysed in the presence of proteinase-K (Sigma Aldrich 31158) at 56°C, then DNA was isolated by silica gel columns purification technology. Quantitative real-time PCR was performed with LightCycler 480 SYBR Green I Master detection kit (Roche 04707516001) on a Light Cycler 480 machine (Roche). Three technical replicates were performed for each sample. Fluorescence curve analysis was performed using LightCycler 480 Software (version 1.5.1.62, Roche). Primer sequences were the following: ND1-Fw: 5'-CCCTACTTCTAACCTCCCTGTTCTTAT-3' ND1-Rv: 5'-CAT-AGGAGGTGTATGAGTTGGTCGTGA-3' ND5-Fw: 5'-ATTTTATTTCTCCAACATACTCGGATT-3' ND5-Rv: 5'-GGGCAGGTTTTGGCTCGTA-3' 18S-Fw: 5'-AGTCGGAGGTTTCGAAGACGAT-3' 18S-Rv: 5'-GCGGGTCATGGGAA-TAACG-3'.

Lactate assay

0.5-2 × 10⁶ cells have been washed and resuspended in 500 µl of motility medium in 96well plate for 4 h. Then, cells have been collected and lactate has been measured by using Lactate Assay Kit (Sigma MAK064), following manufacturer instructions. Measurements were performed using CLARIOstar instrument (BMG Labtech, software version 5.6.1).

High-resolution respirometry with Oroboros device

The oxygen consumption rate (OCR) was measured in the *motility medium* (see above) by a high-resolution 2 mL glass chamber Oxygraph-2k respirometer (Oroboros). The electrode was calibrated at 37 °C, 100% and 0% oxygen before adding CD8⁺ T cells (2 mL at 2.5 × 10⁶ cells/mL) to each chamber and the flux of O₂ consumption was measured in pmol/s. Oligomycin (0.5 µg/mL) was added to block complex V, as an estimate of the contribution of mitochondrial leakage to overall cellular respiration. Increasing amounts of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were added to measure maximum respiratory capacity. Finally, potassium cyanide (KCN) was added to measure the non-mitochondrial respiration.

Measurement of CO₂ flux

15 × 10⁶ CD₈⁺ T cells were incubated in RPMI 1640 medium (Thermo
Fisher 31870) containing 11 mM [U-¹⁴C]-glucose (0.1 mCi/mmol) or in
RPMI 1640 medium (Thermo Fisher 11879) containing 2 mM [U-¹⁴C]-
glutamine (0.1 mCi/mmol). After 6 h, the media were transferred to a
conical glass vial for CO₂ production measurement. Perchloric acid
was injected into the incubation media through the rubber cap to a
final concentration of 4% (v/v). Benzethonium hydroxide was injected
through the rubber cap into a plastic well suspended above the incu-
bation media. During 1 hour of vigorous shaking at 25 °C, the released
[¹⁴C]-CO₂ was trapped by the benzethonium hydroxide and assessed by
scintillation counting.

Flow cytometry

The following antibodies have been used to stain extracellular proteins (1:100 dilution): BV711 anti-CD45RA (Biolegend 304138, clone HI100), APC anti-CD45RO (Biolegend 304210, clone UCHL1), PE anti-CCR7 (Biolegend 353204, clone G043H7), APC-Cy7 anti-CD27 (Biolegend 302816, clone O323), PE-Cy7 anti-CD95 (Biolegend 305621, clone DX2), CD49D-PE (Biolegend 304303, clone 9F10), CD62L-PerCP (Biolegend 304824, clone DREG-56), CD2-APC (Miltenyi 130-116-253, clone REA972), CCR5-A488 (Biolegend 359103, clone J418F1), CXCR3-PE (Biolegend 353706, clone G025H7), CXCR4-PerCP (Biolegend 306515, clone 12G5), CD44-A647 (Biolegend 397512, clone C44-Mab5), CD103-APC (Biolegend 350216, clone Ber-ACT8), cd11a-PE/Cy5 (Biolegend

301210, clone HI111), CCR3-PE (Biolegend 310705, clone 5E8), LPAM1-APC (eBioscience 17-5887-82, clone DATK-32), GLUT1-PE (R&D System FAB1418P, clone 202915). True-Nuclear Transcription Factor Buffer Set (Biolegend 424401) was used to stain intracellular proteins, detected with the following anti-human antibodies (1:50 dilution): PE-anti-GrzB (Biolegend 372208, clone QA16A02), PE-Cy7-anti-Perforin (Biolegend 353315, clone B-D48), APC-anti-IFNγ (Biolegend 502511, clone 4 S.B3), BV421-anti-TNF (Biolegend 502932, clone Mab11), Alexa647-anti-ATP5a (Abcam ab196198, clone EPR13030(B)), PE-anti-IDH2 (Abcam ab212122, clone EPR7577), Alexa488-anti-HK1 (Abcam ab184818, clone EPR10134(B)), Alexa488-anti-PFK2/PFKFB3 (Abcam ab203984, clone EPR12594), Alexa405-anti-GLUT1 (Abcam ab210438, clone EPR3915), DyLight405-anti-G6PD (BioTechnne NBP2-22125V, clone 2H7), anti-MCT1 (BioTechnne FAB8275T, clone 882616). Primary antibodies were incubated o.n. at 4 °C. To evaluate GranzymeB, Perforin, IFNγ and TNF production, 1 × 10⁶ cells have been restimulated for 5 h in a 96well plate with 3 µl of TransAct (Miltenyi 130-111-160). 5 µg/ml BrefeldinA (Biolegend 420601) has been added for the last 2 h and a half. For T cell subpopulations, the following subsets were defined by flow cytometry gating strategy: stem cell memory-like (Tscm, CD45ROnegCD45RAposCD27posCCR7posCD95pos); naïve-like (naive, CD45ROnegCD45RAposCD27posCCR7posCD95neg); effector-like (Teff, CD45ROnegCD45RAposCD27negCCR7neg); central memory-like (Tcm, CD45ROposCD45RAnegCD27posCCR7pos); effector memory-like (Tem, CD45ROposCD45RAnegCD27negCCR7neg). Cells outside the indicated gates were defined as “others”. Gating strategy is reported as supplementary file (Supplementary Fig. 13). To evaluate glucose uptake, mitochondrial membrane potential (MMP), mitochondrial mass or mitochondrial ROS (mtROS), cells were incubated for 20/30 min at 37 °C in the presence of 30 µM 2-NBDG (Thermo NI3195), 100 nM TMRE (Thermo Fisher T669), 100 nM MitoTrackerGreen (Thermo Fisher M7514) or 2.5 µM MitoSox (Thermo Fisher 11579096), respectively. Then, cells have been washed and analysed. Mitochondrial mass (MitoMass) has been calculated as (MitoTrackerGreen/FSC) ratio. MMP has been calculated as (TMRE/MitoTrackerGreen) ratio, mtROS amount as (MitoSox/FSC) ratio. Samples were analysed using BD Accuri C6 (CFlow Plus software v1.0.264.15) or BD Fortessa (FACSDive software v6.1.3).

Infiltration into collagen gel

5 × 10⁵ human BxPC3 tumor cells were resuspended into 450 µl of complete RPMI medium plus neutralized collagen solution (2 mg/ml) and inserted into one side chamber of µ-slide Chemotaxis Chamber (Ibidi 80326). The other side chamber was filled with medium alone. After 24 h, T cells were stained with Calcein Green or Calcein Red as described above, mixed in a 1:1 ratio, and 2 × 10⁶ cells were resuspended into 80 µl and inserted into the middle chamber of the µ-slide. Z-stack images were acquired at 0 h and 2 h. ImageJ software was used to identify cells (as ROIs) and to calculate the minimum distance from starting area.

Anti-EGFR CAR and PercevalHR lentiviral infection

To generate anti-EGFR CAR, downstream of a signal peptide scFv
sequences derived from nimotuzumab Ab (IMGT/2Dstructure-DB INN
8545H, 8545 L) were introduced into the expression cassette encoding
CD8 hinge and transmembrane domains (194-248 Aa, GenBank:
AAH25715.1), 4-1BB costimulatory domain (214-255 Aa, GenBank:
AAX42660.1), CD3z signaling domain (52-163 Aa, GenBank:
NP_000725.1) and GFP coding sequence (downstream of IRES
sequence). Production of lentiviral particles was performed by tran-
sient co-transfection (polyethylenimine:DNA at 3.5:1 ratio) of HEK
293 T cells with the second-generation packaging system plasmid
psPAX2 and pMD2.G (Addgene, 12260 and 12259). Viral supernatants
were harvested at 48 h post-transfection. Viral particles were con-
centrated by ultracentrifugation at 25.000 g for 2 h and conserved at
GTGG-3' (Hs.Cas9.OGDH.1.AB) 5'-ACGAATGCCGGAGCCCCACCGG-3'
(Hs.Cas9.OGDH.1.AC).

qRT-PCR for mitochondrial DNA

DNA extraction was performed with FastPure Blood/Cell/Tissue/Bacteria DNA Isolation Mini Kit (Vazyme Biotech DC112-01). Cells were lysed in the presence of proteinase-K (Sigma Aldrich 31158) at 56°C, then DNA was isolated by silica gel columns purification technology. Quantitative real-time PCR was performed with LightCycler 480 SYBR Green I Master detection kit (Roche 04707516001) on a Light Cycler 480 machine (Roche). Three technical replicates were performed for each sample. Fluorescence curve analysis was performed using LightCycler 480 Software (version 1.5.1.62, Roche). Primer sequences were the following: ND1-Fw: 5'-CCCTACTTCTAACCTCCCTGTTCTTAT-3' ND1-Rv: 5'-CAT-AGGAGGTGTATGAGTTGGTCGTGA-3' ND5-Fw: 5'-ATTTTATTTCTCCAACATACTCGGATT-3' ND5-Rv: 5'-GGGCAGGTTTTGGCTCGTA-3' 18S-Fw: 5'-AGTCGGAGGTTTCGAAGACGAT-3' 18S-Rv: 5'-GCGGGTCATGGGAA-TAACG-3'.

Lactate assay

0.5-2 × 10⁶ cells have been washed and resuspended in 500 µl of motility medium in 96well plate for 4 h. Then, cells have been collected and lactate has been measured by using Lactate Assay Kit (Sigma MAK064), following manufacturer instructions. Measurements were performed using CLARIOstar instrument (BMG Labtech, software version 5.6.1).

High-resolution respirometry with Oroboros device

The oxygen consumption rate (OCR) was measured in the *motility medium* (see above) by a high-resolution 2 mL glass chamber Oxygraph-2k respirometer (Oroboros). The electrode was calibrated at 37 °C, 100% and 0% oxygen before adding CD8⁺ T cells (2 mL at 2.5 × 10⁶ cells/mL) to each chamber and the flux of O₂ consumption was measured in pmol/s. Oligomycin (0.5 µg/mL) was added to block complex V, as an estimate of the contribution of mitochondrial leakage to overall cellular respiration. Increasing amounts of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were added to measure maximum respiratory capacity. Finally, potassium cyanide (KCN) was added to measure the non-mitochondrial respiration.

Measurement of CO₂ flux

15 × 10⁶ CD₈⁺ T cells were incubated in RPMI 1640 medium (Thermo
Fisher 31870) containing 11 mM [U-¹⁴C]-glucose (0.1 mCi/mmol) or in
RPMI 1640 medium (Thermo Fisher 11879) containing 2 mM [U-¹⁴C]-
glutamine (0.1 mCi/mmol). After 6 h, the media were transferred to a
conical glass vial for CO₂ production measurement. Perchloric acid
was injected into the incubation media through the rubber cap to a
final concentration of 4% (v/v). Benzethonium hydroxide was injected
through the rubber cap into a plastic well suspended above the incu-
bation media. During 1 hour of vigorous shaking at 25 °C, the released
[¹⁴C]-CO₂ was trapped by the benzethonium hydroxide and assessed by
scintillation counting.

Flow cytometry

The following antibodies have been used to stain extracellular proteins (1:100 dilution): BV711 anti-CD45RA (Biolegend 304138, clone HI100), APC anti-CD45RO (Biolegend 304210, clone UCHL1), PE anti-CCR7 (Biolegend 353204, clone G043H7), APC-Cy7 anti-CD27 (Biolegend 302816, clone O323), PE-Cy7 anti-CD95 (Biolegend 305621, clone DX2), CD49D-PE (Biolegend 304303, clone 9F10), CD62L-PerCP (Biolegend 304824, clone DREG-56), CD2-APC (Miltenyi 130-116-253, clone REA972), CCR5-A488 (Biolegend 359103, clone J418F1), CXCR3-PE (Biolegend 353706, clone G025H7), CXCR4-PerCP (Biolegend 306515, clone 12G5), CD44-A647 (Biolegend 397512, clone C44-Mab5), CD103-APC (Biolegend 350216, clone Ber-ACT8), cd11a-PE/Cy5 (Biolegend

301210, clone HI111), CCR3-PE (Biolegend 310705, clone 5E8), LPAM1-APC (eBioscience 17-5887-82, clone DATK-32), GLUT1-PE (R&D System FAB1418P, clone 202915). True-Nuclear Transcription Factor Buffer Set (Biolegend 424401) was used to stain intracellular proteins, detected with the following anti-human antibodies (1:50 dilution): PE-anti-GrzB (Biolegend 372208, clone QA16A02), PE-Cy7-anti-Perforin (Biolegend 353315, clone B-D48), APC-anti-IFNγ (Biolegend 502511, clone 4 S.B3), BV421-anti-TNF (Biolegend 502932, clone Mab11), Alexa647-anti-ATP5a (Abcam ab196198, clone EPR13030(B)), PE-anti-IDH2 (Abcam ab212122, clone EPR7577), Alexa488-anti-HK1 (Abcam ab184818, clone EPR10134(B)), Alexa488-anti-PFK2/PFKFB3 (Abcam ab203984, clone EPR12594), Alexa405-anti-GLUT1 (Abcam ab210438, clone EPR3915), DyLight405-anti-G6PD (BioTechnne NBP2-22125V, clone 2H7), anti-MCT1 (BioTechnne FAB8275T, clone 882616). Primary antibodies were incubated o.n. at 4 °C. To evaluate GranzymeB, Perforin, IFNγ and TNF production, 1 × 10⁶ cells have been restimulated for 5 h in a 96well plate with 3 µl of TransAct (Miltenyi 130-111-160). 5 µg/ml BrefeldinA (Biolegend 420601) has been added for the last 2 h and a half. For T cell subpopulations, the following subsets were defined by flow cytometry gating strategy: stem cell memory-like (Tscm, CD45ROnegCD45RAposCD27posCCR7posCD95pos); naïve-like (naive, CD45ROnegCD45RAposCD27posCCR7posCD95neg); effector-like (Teff, CD45ROnegCD45RAposCD27negCCR7neg); central memory-like (Tcm, CD45ROposCD45RAnegCD27posCCR7pos); effector memory-like (Tem, CD45ROposCD45RAnegCD27negCCR7neg). Cells outside the indicated gates were defined as "others". Gating strategy is reported as supplementary file (Supplementary Fig. 13). To evaluate glucose uptake, mitochondrial membrane potential (MMP), mitochondrial mass or mitochondrial ROS (mtROS), cells were incubated for 20/30 min at 37 °C in the presence of 30 µM 2-NBDG (Thermo NI3195), 100 nM TMRE (Thermo Fisher T669), 100 nM MitoTrackerGreen (Thermo Fisher M7514) or 2.5 µM MitoSox (Thermo Fisher 11579096), respectively. Then, cells have been washed and analysed. Mitochondrial mass (MitoMass) has been calculated as (MitoTrackerGreen/FSC) ratio. MMP has been calculated as (TMRE/MitoTrackerGreen) ratio, mtROS amount as (MitoSox/FSC) ratio. Samples were analysed using BD Accuri C6 (CFlow Plus software v1.0.264.15) or BD Fortessa (FACSDive software v6.1.3).

Infiltration into collagen gel

5 × 10⁵ human BxPC3 tumor cells were resuspended into 450 µl of complete RPMI medium plus neutralized collagen solution (2 mg/ml) and inserted into one side chamber of µ-slide Chemotaxis Chamber (Ibidi 80326). The other side chamber was filled with medium alone. After 24 h, T cells were stained with Calcein Green or Calcein Red as described above, mixed in a 1:1 ratio, and 2 × 10⁶ cells were resuspended into 80 µl and inserted into the middle chamber of the µ-slide. Z-stack images were acquired at 0 h and 2 h. ImageJ software was used to identify cells (as ROIs) and to calculate the minimum distance from starting area.

Anti-EGFR CAR and PercevalHR lentiviral infection

To generate anti-EGFR CAR, downstream of a signal peptide scFv
sequences derived from nimotuzumab Ab (IMGT/2Dstructure-DB INN
8545H, 8545 L) were introduced into the expression cassette encoding
CD8 hinge and transmembrane domains (194-248 Aa, GenBank:
AAH25715.1), 4-1BB costimulatory domain (214-255 Aa, GenBank:
AAX42660.1), CD3z signaling domain (52-163 Aa, GenBank:
NP_000725.1) and GFP coding sequence (downstream of IRES
sequence). Production of lentiviral particles was performed by tran-
sient co-transfection (polyethylenimine:DNA at 3.5:1 ratio) of HEK
293 T cells with the second-generation packaging system plasmid
psPAX2 and pMD2.G (Addgene, 12260 and 12259). Viral supernatants
were harvested at 48 h post-transfection. Viral particles were con-
centrated by ultracentrifugation at 25.000 g for 2 h and conserved at
-80 °C. Anti-EGFR CD8⁺ T cells (defined as CD8⁺CAR) were generated by infecting 6 × 10⁵ CD8⁺ T cells activated for 72 h with 50 µL (MOI2) of lentiviral particles in 600 µL of RPMI complete medium (supplemented with IL-7 + IL-15) in 48 well plate. After 3 days, cells have been collected, washed, and used for subsequent analysis. PercevalHR plasmid<sup>39,40</sup> (Addgene 49083) was used to generate lentiviral particles using the same procedure as for “CAR” particles. After 1 week of stimulation, cells were stained with Calcein Red, and motility was analysed in 2 mg/ml collagen gel. PercevalHR and Calcein Red signals was measured at timepoint 0 and then the ratio was calculated. Cells were defined as motile or immotile by visual inspection following motility analysis of the TrackMate software.

## Cytotoxicity assay

Anti-EGFR CAR T cell cytotoxicity has been evaluated using mCherry<sup>pos</sup> A549 cells. Briefly, A549 cells were plated at 1 × 10⁵ cells/250 µL on µ-Slide 8 Well slides (Ibidi 80826) in DMEM medium. After 24 h (time 0 h), anti-EGFR CAR T cells were added to obtain a CAR:target ratio of 2.5:1, 5:1 or 10:1 in RPMI complete medium. A549 cell viability was evaluated by quantifying mCherry fluorescence (MFI) at different time points (24 h, 48 h) with a fluorescent microscope and normalized to MFI values in absence of CAR T cells.

## Orthotopic and subcutaneous tumor models

For the orthotopic tumor model, 2 × 10⁶ human A549 cells (mCherry⁺Luciferase⁺) were injected i.v. into the tail vein of 2 months-old NSG nude mice (independently of sex). After 3 weeks, mice were either (i) sacrificed to obtain lung slices to be used in motility experiments (see above) or (ii) infused i.v. with 5 × 10⁶ anti-EGFR CD8⁺ CAR T cells into the tail vein. After 4 days, mice were sacrificed, and lungs were collected. For flow cytometry, lungs were digested with DNase (Roche 4536282001) and Liberase (Roche 5401119001) solution at 37 °C for 1 h and then smashed using a 70 µm Cell Strainer filter (Corning 352350). Collected cells were stained with Alexa647-anti-CD8 (Biolegend 344726) and PE-anti-EpCAM (Biolegend 324206) Abs for flow cytometry analysis (BD Accuri C6). For immunofluorescence, lungs were fixed in PLP solution (see above) for 24 h at 4 °C and, cut into 1-2mm³ pieces, embedded into 7% agarose (Sigma Aldrich A0701), and sliced using a vibratome (VT 1000 S, Leica) to obtain 150 µm-thick sections. Slices were processed using FITC-anti-CD3 (Biolegend 344804) and PE-anti-EPCAM (Biolegend 324206) Abs. To follow tumor growth, 3 days after i.v. injection of A549 cells, mice were injected i.v. with 5 × 10⁶ anti-EGFR CD8⁺ CAR T cells into the tail vein. Tumor growth in the lungs was followed by bioluminescence using PhotonIMAGER system (Biospacelab) and analysis was performed using M3Vision software (Biospacelab, software version 1.2.1.32016). To assess tumor burden in the orthotopic model, an evaluation grid was implemented according to our ethical committee in order to sacrifice mice reaching a defined endpoint threshold (calculated by taking into account tumor bioluminescence score, mouse behavior, physical appearance, weight, etc.). None of the mice reached the endpoint threshold score at the end of the experiment. For the subcutaneous tumor model, 5 × 10⁶ human A549 cells were injected s.c. into the right flank of 2 months-old NSG nude mice (independently of sex). To evaluate CAR T cell infiltration, mice were injected i.v. after 5/6 weeks with 5 × 10⁶ anti-EGFR CD8⁺ CAR T cells and sacrificed 4 days after to perform the same analyses described for orthotopic tumors. Alternatively, 10 days after A549 cell injection, A549-derived tumor-bearing mice were i.v. inoculated with 5 × 10⁶ anti-EGFR CD8⁺ CAR T cells and tumor growth was followed through caliper measurement for additional 4 weeks. Then, mice were sacrificed, tumors were digested (like what described above for lungs), and collected cells were stained with PE-anti-EPCAM (Biolegend 324206) and Alexa488-anti-CD45 (Biolegend 304017) Abs for flow cytometry analysis (BD Accuri C6). We did not exceed the maximal s.c. tumor size permitted by our ethics committee (1500 mm³).

For the competition assay, anti-EGFR ctrl and Rapa CD8⁺ CAR T cells were stained with 1 µM CFSE (Invitrogen C34554) or 5 µM Cell Proliferation Dye eFluor670 (eBioscience 65-0840-90) and then mixed in a 1:1 ratio before being injected into tumor-bearing NSG mice (lung or s.c. tumor models as indicated before). After 3-4 days, the mice were sacrificed and infiltration into lung or s.c. tumors was assessed by flow cytometry (only for the s.c. model) or immunofluorescence on vibratome-cut slices using a spinning-disk (Yokogawa CSU-X1) Leica DM6000FS microscope equipped with a 37 °C heated-chamber, an ORCA-Flash 4.0 camera (CI1440 Hamamatsu) and Metamorph software (version 7.8.9, Molecular Devices).

Subcutaneous tumors were also obtained through injection of human 5 × 10⁶ BxPC3 cells s.c. into the right flank of 2 months-old NSG nude mice (independently of sex). After 3/4 weeks, mice were sacrificed, and tumors were collected to obtain viable tumor slices used in the slice assay (see above). We did not exceed the maximal s.c. tumor size permitted by our ethics committee (1500 mm³).

## Western blot

Western blots were performed as previously described in ref. 79. Briefly, cells have been lysed in RIPA buffer 20 min on ice (Tris-HCl 50 mM pH8, NaCl 150 mM, Nonidet-P40 1%, Sodium deoxycolate 0.5%, SDS 0.1%) supplemented with Protease Inhibitor Cocktail (Thermo Fisher 78429) and 1 mM NaVO₄. Samples were then centrifuged at 4 °C 20 min and denatured 5 min at 95 °C in denaturation buffer (with 5% beta-mercaptoethanol). Samples were run on 4–12% polyacrylamide gels (Invitrogen NW04125BOX) and transferred on PVDF membranes (Thermo Fisher 88518) using Mini-Trans Blot Cell (Bio-Rad 1703930). Membranes were blocked in 3% w/v non-fat dry milk containing 0.1% Tween20 (Sigma-Aldrich) and incubated with the appropriate antibodies o.n. at 4 °C. The following primary anti-human antibodies have been used (1:1000 dilution): anti-actin (Cell Signaling 4970, clone 13E5), anti-OGDH (Sigma Aldrich HPA019514), anti-Hsp90 (Cell Signaling 4877 S, clone C45G5), anti-PDHA1 + A2 (Cell Signaling 2784 S) and OxPhos Human WB Antibody Cocktail (Thermo Fisher 45-8199). Detection of protein signals was performed using ECL Prime Western Blotting Detection Reagent (Cytiva RPN2236) and Fusion FX instrument (Vilber Lourmat, software version 16.16.0.0). The original scans of the western blots presented in the manuscript are reported as supplementary file (Supplementary Fig. 14).

## Transendothelial migration assay

5µm-pore transwell inserts (Costar 3421) have been coated for 1 h at 37 °C with gelatin (Gibco S006100) and 10 µg/ml bovine fibronectin protein (Thermo Fisher 33010018). Subsequently, 5 × 10⁵ HUVEC cells were added into the insert, let expanded for 24 h and stimulated o.n. with 10 ng/ml human TNF (Thermo Fisher PHC3016). The day after, CAR T cells were stained with either Calcein Green or Cell Proliferation Dye eFluor670 (Thermo Fisher 65-0840-85) (same procedure used for motility assay, see above) and mixed in a 1:1 ratio. Then, 2 × 10⁵ cells were added to the transwell upper chamber in a motility medium (BSA, no FBS). Transwell lower chamber was filled with RPMI complete medium (10% FBS). The transmigrated cells were collected and quantified by BD Accuri C6 cytometer.

## Statistical analysis

In the Figure legends, “n” indicates either the number of biological independent samples, donors, the number of movies (no more than 3 from the same donor) or the number of mice used. Data are expressed as mean ± SEM or as box and whiskers plot. Comparisons between normal groups were done using paired or unpaired two-tailed Student's T-test (two groups) or One-way and Two-way ANOVA. Mann-Whitney Rank Sum Test (unpaired), Wilcoxon Signed Rank Test (paired) or ANOVA on Ranks were used if samples did not meet assumptions of normality. Adjustments for pairwise comparisons were
−80 °C. Anti-EGFR CD8⁺ T cells (defined as CD8⁺CAR) were generated by infecting 6 × 10⁵ CD8⁺ T cells activated for 72 h with 50 µL (MOI2) of lentiviral particles in 600 µL of RPMI complete medium (supplemented with IL-7 + IL-15) in 48 well plate. After 3 days, cells have been collected, washed, and used for subsequent analysis. PercevalHR plasmid<sup>39,40</sup> (Addgene 49083) was used to generate lentiviral particles using the same procedure as for “CAR” particles. After 1 week of stimulation, cells were stained with Calcein Red, and motility was analysed in 2 mg/ ml collagen gel. PercevalHR and Calcein Red signals was measured at timepoint 0 and then the ratio was calculated. Cells were defined as motile or immotile by visual inspection following motility analysis of the TrackMate software.

Cytotoxicity assay

Anti-EGFR CAR T cell cytotoxicity has been evaluated using mCherry<sup>pos</sup> A549 cells. Briefly, A549 cells were plated at 1 × 10<sup>5</sup> cells/250 µl on µ-Slide 8 Well slides (Ibidi 80826) in DMEM medium. After 24 h (time 0 h), anti-EGFR CAR T cells were added to obtain a CAR:target ratio of 2.5:1, 5:1 or 10:1 in RPMI complete medium. A549 cell viability was evaluated by quantifying mCherry fluorescence (MFI) at different time points (24 h, 48 h) with a fluorescent microscope and normalized to MFI values in absence of CAR T cells.

Orthotopic and subcutaneous tumor models

For the orthotopic tumor model, 2 × 10⁶ human A549 cells (mCherry⁺Luciferase⁺) were injected i.v. into the tail vein of 2 months-old NSG nude mice (independently of sex). After 3 weeks, mice were either (i) sacrificed to obtain lung slices to be used in motility experiments (see above) or (ii) infused i.v. with 5 × 10⁶ anti-EGFR CD8⁺ CAR T cells into the tail vein. After 4 days, mice were sacrificed, and lungs were collected. For flow cytometry, lungs were digested with DNase (Roche 4536282001) and Liberase (Roche 5401119001) solution at 37 °C for 1 h and then smashed using a 70 µm Cell Strainer filter (Corning 352350). Collected cells were stained with Alexa647-anti-CD8 (Biolegend 344726) and PE-anti-EpCAM (Biolegend 324206) Abs for flow cytometry analysis (BD Accuri C6). For immunofluorescence, lungs were fixed in PLP solution (see above) for 24 h at 4 °C and, cut into 1-2mm³ pieces, embedded into 7% agarose (Sigma Aldrich A0701), and sliced using a vibratome (VT 1000 S, Leica) to obtain 150µm-thick sections. Slices were processed using FITC-anti-CD3 (Biolegend 344804) and PE-anti-EPCAM (Biolegend 324206) Abs. To follow tumor growth, 3 days after i.v. injection of A549 cells, mice were injected i.v. with 5 × 10⁵ anti-EGFR CD8⁺ CAR T cells into the tail vein. Tumor growth in the lungs was followed by bioluminescence using PhotonIMAGER system (Biospacelab) and analysis was performed using M3Vision software (Biospacelab, software version 1.2.1.32016). To assess tumor burden in the orthotopic model, an evaluation grid was implemented according to our ethical committee in order to sacrifice mice reaching a defined endpoint threshold (calculated by taking into account tumor bioluminescence score, mouse behavior, physical appearance, weight, etc.). None of the mice reached the endpoint threshold score at the end of the experiment. For the subcutaneous tumor model, 5 × 10⁶ human A549 cells were injected s.c. into the right flank of 2 months-old NSG nude mice (independently of sex). To evaluate CAR T cell infiltration, mice were injected i.v. after 5/6 weeks with 5 × 10⁶ anti-EGFR CD8⁺ CAR T cells and sacrificed 4 days after to perform the same analyses described for orthotopic tumors. Alternatively, 10 days after A549 cell injection, A549-derived tumor-bearing mice were i.v. inoculated with 5 × 10⁶ anti-EGFR CD8⁺ CAR T cells and tumor growth was followed through caliper measurement for additional 4 weeks. Then, mice were sacrificed, tumors were digested (like what described above for lungs), and collected cells were stained with PE-anti-EPCAM (Biolegend 324206) and Alexa488-anti-CD45 (Biolegend 304017) Abs for flow cytometry analysis (BD Accuri C6). We did not exceed the maximal s.c. tumor size permitted by our ethics committee (1500 mm³).

For the competition assay, anti-EGFR ctrl and Rapa CD8⁺ CAR T cells were stained with 1 µM CFSE (Invitrogen C34554) or 5 µM Cell Proliferation Dye eFluor670 (eBioscience 65-0840-90) and then mixed in a 1:1 ratio before being injected into tumor-bearing NSG mice (lung or s.c. tumor models as indicated before). After 3–4 days, the mice were sacrificed and infiltration into lung or s.c. tumors was assessed by flow cytometry (only for the s.c. model) or immunofluorescence on vibratome-cut slices using a spinning-disk (Yokogawa CSU-X1) Leica DM6000FS microscope equipped with a 37 °C heated-chamber, an ORCA-Flash 4.0 camera (CI1440 Hamamatsu) and Metamorph software (version 7.8.9, Molecular Devices).

Subcutaneous tumors were also obtained through injection of human 5 × 10⁶ BxPC3 cells s.c. into the right flank of 2 months-old NSG nude mice (independently of sex). After 3/4 weeks, mice were sacrificed, and tumors were collected to obtain viable tumor slices used in the slice assay (see above). We did not exceed the maximal s.c. tumor size permitted by our ethics committee (1500 mm³).

Western blot

Western blots were performed as previously described in ref. 79. Briefly, cells have been lysed in RIPA buffer 20 min on ice (Tris-HCl 50 mM pH8, NaCl 150 mM, Nonidet-P40 1%, Sodium deoxycolate 0.5%, SDS 0.1%) supplemented with Protease Inhibitor Cocktail (Thermo Fisher 78429) and 1 mM NaVO₄. Samples were then centrifuged at 4 °C 20 min and denatured 5 min at 95 °C in denaturation buffer (with 5% beta-mercaptoethanol). Samples were run on 4–12% polyacrylamide gels (Invitrogen NW04125BOX) and transferred on PVDF membranes (Thermo Fisher 88518) using Mini-Trans Blot Cell (Bio-Rad 1703930). Membranes were blocked in 3% w/v non-fat dry milk containing 0.1% Tween20 (Sigma-Aldrich) and incubated with the appropriate antibodies o.n. at 4 °C. The following primary anti-human antibodies have been used (1:1000 dilution): anti-actin (Cell Signaling 4970, clone 13E5), anti-OGDH (Sigma Aldrich HPA019514), anti-Hsp90 (Cell Signaling 4877 S, clone C45G5), anti-PDHA1 + A2 (Cell Signaling 2784 S) and OxPhos Human WB Antibody Cocktail (Thermo Fisher 45-8199). Detection of protein signals was performed using ECL Prime Western Blotting Detection Reagent (Cytiva RPN2236) and Fusion FX instrument (Vilber Lourmat, software version 16.16.0.0). The original scans of the western blots presented in the manuscript are reported as supplementary file (Supplementary Fig. 14).

Transendothelial migration assay

5µm-pore transwell inserts (Costar 3421) have been coated for 1 h at 37 °C with gelatin (Gibco S006100) and 10 µg/ml bovine fibronectin protein (Thermo Fisher 33010018). Subsequently, 5 × 10⁵ HUVEC cells were added into the insert, let expanded for 24 h and stimulated o.n. with 10 ng/ml human TNF (Thermo Fisher PHC3016). The day after, CAR T cells were stained with either Calcein Green or Cell Proliferation Dye eFluor670 (Thermo Fisher 65-0840-85) (same procedure used for motility assay, see above) and mixed in a 1:1 ratio. Then, 2 × 10⁵ cells were added to the transwell upper chamber in a motility medium (BSA, no FBS). Transwell lower chamber was filled with RPMI complete medium (10% FBS). The transmigrated cells were collected and quantified by BD Accuri C6 cytometer.

Statistical analysis

In the Figure legends, “n” indicates either the number of biological independent samples, donors, the number of movies (no more than 3 from the same donor) or the number of mice used. Data are expressed as mean ± SEM or as box and whiskers plot. Comparisons between normal groups were done using paired or unpaired two-tailed Student's T-test (two groups) or One-way and Two-way ANOVA. Mann-Whitney Rank Sum Test (unpaired), Wilcoxon Signed Rank Test (paired) or ANOVA on Ranks were used if samples did not meet assumptions of normality. Adjustments for pairwise comparisons were
performed using post hoc tests. Statistical analysis has been performed using SigmaPlot (v15).

## Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

## Data availability

Raw immunofluorescence images of NSCLC samples generated in this study have been deposited on Zenodo with accession number [10.57889] and DOI number (https://doi.org/10.5788/Simula.et.al.2023) with unrestricted access. All other data supporting the findings of this work are available in the main text, the Supplementary Information, and Source Data files. Source data are provided with this paper.

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## Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

## Data availability

Raw immunofluorescence images of NSCLC samples generated in this study have been deposited on Zenodo with accession number [10.57889] and DOI number (https://doi.org/10.5788/Simula.et.al.2023) with unrestricted access. All other data supporting the findings of this work are available in the main text, the Supplementary Information, and Source Data files. Source data are provided with this paper.

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## Acknowledgements

This work was partially supported by the European Union's Horizon 2020 research and innovation programme under the Marie-Skłodowska Curie grant agreement No. 945298 "ParisRegionFP" (LS), the French "Ligue Nationale contre le Cancer" (Equipes labellisées, ED), GeFluc Ile-De-France (Call 2021 & Call 2023, LS), European Union's Horizon Europe programme (HE-MSCA-PF 101062302, LS), and Fondation ARC (PDF2 ARCPDF222020010001123, LS). LS and MCAG were also supported by a grant from Institut Cochin (PIC 2022). MF and DA were respectively supported by "Ligue Nationale contre le Cancer" and "China Scholarship
Council" PhD fellowships. We are extremely thankful to members of the Cochin Institute Imaging (IMAG'IC), Cytometry and ImmunoBiology (CYBIO) and In vivo Imaging (PIV) facilities for their help and support in performing microscopy, flow cytometry and animal experiments, respectively.

**Author contributions**

Conceptualization, Funding Acquisition & Writing - Original Draft: L.S. and E.D. Investigation: L.S., M.C.-A.G., M.F., D.A., F.P., G.B., N.B., A.R. and I.R. Formal Analysis: L.V. Resources: D.D., A.L.M., M.A., P.I., D.A. and F.P. Writing - Review & Editing: M.C.-A.G., F.P. and P.I. All authors approved the manuscript.

**Competing interests**

The authors declare no competing interests.

**Additional information**

**Supplementary information** The online version contains supplementary material available at https://doi.org/10.1038/s41467-024-46377-7.

**Correspondence** and requests for materials should be addressed to Luca Simula or Emmanuel Donnadieu.

**Peer review information** *Nature Communications* thanks Minsoo Kim, Roddy O'Connor and Ingunn Stromnes for their contribution to the peer review of this work. A peer review file is available.

**Reprints and permissions information** is available at http://www.nature.com/reprints

**Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

© The Author(s) 2024
Council" PhD fellowships. We are extremely thankful to members of the Cochin Institute Imaging (IMAG'IC), Cytometry and ImmunoBiology (CYBIO) and In vivo Imaging (PIV) facilities for their help and support in performing microscopy, flow cytometry and animal experiments, respectively.

**Author contributions**

Conceptualization, Funding Acquisition & Writing - Original Draft: L.S. and E.D. Investigation: L.S., M.C.-A.G., M.F., D.A., F.P., G.B., N.B., A.R. and I.R. Formal Analysis: L.V. Resources: D.D., A.L.M., M.A., P.I., D.A. and F.P. Writing - Review & Editing: M.C.-A.G., F.P. and P.I. All authors approved the manuscript.

**Competing interests**

The authors declare no competing interests.

**Additional information**

**Supplementary information** The online version contains supplementary material available at https://doi.org/10.1038/s41467-024-46377-7.

**Correspondence** and requests for materials should be addressed to Luca Simula or Emmanuel Donnadieu.

**Peer review information** *Nature Communications* thanks Minsoo Kim, Roddy O'Connor and Ingunn Stromnes for their contribution to the peer review of this work. A peer review file is available.

**Reprints and permissions information** is available at http://www.nature.com/reprints

**Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

© The Author(s) 2024