Metabolic flux profiling of MDCK cells during growth and canine

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1 Metabolic flux profiling of MDCK cells during growth and canine adenovirus vector production Nuno Carinhas 1#, Daniel AM Pais 1,2#, Alexey Koshkin 1,2, Paulo Fernandes 1,2,, Ana S Coroadinha 1,2, Manuel JT Carrondo 1,2, Paula M Alves 1,2, Ana P Teixeira 1,2* 1 ibet, Instituto de Biologia Experimental e Tecnológica, Apartado 12, Oeiras, Portugal; 2 Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, Oeiras, Portugal # Equal contribution Current address: Autolus, London, UK *Corresponding author: anat@itqb.unl.pt

2 Supplementary information: complete metabolic network model used for nonstationary 13 C-MFA. Glycolysis R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 G6P (abcdef) F6P (abcdef) F6P (abcdef) FBP (abcdef) FBP (abcdef) DHAP (cba) + GAP (def) DHAP (abc) GAP (abc) GAP (abc) 3PG (abc) 3PG (abc) PEP (abc) PEP (abc) Pyr.c (abc) Pentose-phosphate pathway G6P (abcdef) P5P (bcdef) + CO 2 (a) P5P (abcde) + P5P (pqrst) GAP (rst) + S7P (pqabcde) S7P (abcdefg) + GAP (xyz) E4P (defg) + F6P (abcxyz) E4P (abcd) + P5P (pqrst) GAP (rst) + F6P (pqabcd) Lactate and alanine accumulation Pyr.c (abc) Lac (abc) Pyr.c (abc) Ala (abc) TCA cycle and pyruvate cycling Pyr.m (abc) AcCoA.m (bc) + CO 2 (a) Pyr.m (abc) + CO 2 (d) OAA (abcd) OAA (abcd) + AcCoA.m (ef) Cit (dcbfea) Cit (abcdef) AKG (abcde) + CO 2 (f) AKG (abcde) SucCoA (bcde) + CO 2 (a) SucCoA (abcd) Suc (abcd) Suc (abcd) Fum (abcd) Fum (abcd) Mal (abcd) Mal (abcd) OAA (abcd) Mal (abcd) Pyr.m (abc) + CO 2 (d) Lipid precursor generation Cit (dcbfea) OAA (abcd) + AcCoA.c (ef) Amino acids metabolism Gln (abcde) Glu (abcde) AKG (abcde) Glu (abcde) Asn (abcd) Asp (abcd) Asp (abcd) OAA (abcd) 3PG (abc) Ser (abc) Ser (abc) Pyr.c (abc) Ser (abc) Gly (ab) + C1 (c) Glu (abcde) Pro (abcde) Val (abcde) + CO 2 (f) Suc (dcef) + CO 2 (a) + CO 2 (b)

3 R34 R35 R36 R37 R38 R39 R40 R41 R42 R43 R44 Ile (abcdef) + CO 2 (g) Suc (bcdg) + AcCoA.m (ef) + CO 2 (a) Leu (abcdef) + CO 2 (g) AcCoA.m (bc) + AcCoA.m (de) + AcCoA.m (gf) + CO 2 (a) Thr (abcd) AcCoA.m (cd) + Gly (ab) Phe (abcdefghi) Tyr (abcdefghi) Tyr (abcdefghi) Fum (defg) + AcCoA.m (bc) + AcCoA.m (hi) + CO 2 (a) Met (abcde) + Ser (fgh) + CO 2 (i) Suc (bcdi) + Cys.snk (fgh) + CO 2 (a) + C1 (e) Lys (abcdef) CO 2 (a) + CO 2 (f) + AcCoA.m (bc) + AcCoA.m (de) His (abcdef) Glu (edcba) + C1 (f) Arg (abcdef) Glu (abcde) + Urea.snk (f) Glu (abcde) + CO 2 (f) Arg (abcdef) Intracellular transport Pyr.c (abc) Pyr.m (abc) Extracellular transport R45 R46 R47 R48 R49 R50 R51 R52 R53 R54 R55 R56 R57 R58 R59 R60 R61 R62 R63 R64 R65 CO 2 (a) CO 2.ext (a) Glc.ext (abcdef) G6P (abcdef) Lac (abc) Lac.ext (abc) Ala (abc) Ala.ext (abc) Gln.ext (abcde) Gln (abcde) Glu (abcde) Glu.ext (abcde) Asp (abcd) Asp.ext (abcd) Asn (abcd) Asn.ext (abcd) Ser.ext (abc) Ser (abc) Gly (ab) Gly.ext (ab) Pro.ext (abcde) Pro (abcde) Val.ext (abcde) Val (abcde) Ile.ext (abcdef) Ile (abcdef) Leu.ext (abcdef) Leu (abcdef) Thr.ext (abcd) Thr (abcd) Phe.ext (abcdefghi) Phe (abcdefghi) Tyr.ext (abcdefghi) Tyr (abcdefghi) Met.ext (abcde) Met (abcde) Lys.ext (abcdef) Lys (abcdef) His.ext (abcdef) His (abcdef) Arg.ext (abcdef) Arg (abcdef) Biomass formation R66 324*Ala *Glu *Gln *Gly *Ser *Lys *Leu + 175*Ile *Arg *Asp *Thr *Val *Met *Phe *Tyr *His + 169*Pro *Asn *G6P *P5P *C *DHAP *AcCoA.c Biomass Coefficients in the lumped biomass formation reaction represent the nmol content in 10 6 cells. Suffix abbreviations: mitochondrial (.m), cytosolic (.c), extracellular (.ext), sink (.snk). Sink pools were used for metabolites that could not be balanced (Cys, Urea).

4 Supplementary information: list of balanced and unbalanced metabolite pools. Balanced metabolite pools: G6P, F6P, FBP, DHAP, GAP, 3PG, PEP, Pyr.c, Pyr.m, P5P, CO2, S7P, E4P, Lac, Ala, AcCoA.m, AcCoA.c, OAA, Cit, AKG, SucCoA, Suc, Fum, Mal, Gln, Glu, Asn, Asp, Ser, Gly, C1, Pro, Val, Ile, Leu, Thr, Phe, Tyr, Met, Lys, His, Arg. Unbalanced metabolite pools: CO2.ext, Glc.ext, Lac.ext, Ala.ext, Gln.ext, Glu.ext, Asp.ext, Asn.ext, Ser.ext, Gly.ext, Pro.ext, Val.ext, Ile.ext, Leu.ext, Thr.ext, Phe.ext, Tyr.ext, Met.ext, Lys.ext, His.ext, Arg.ext, Cys.snk, Urea.snk, Biomass. Supplementary information: calculation of MID errors for flux estimation. In our experimental design, each MID datapoint corresponds to an independent culture, so that the measured MID dynamic profiles incorporate both analytical and biological variability. However, individual errors for MID measurements could not be experimentally determined. Therefore, a default standard error vector was initially assumed by INCA: 0.3 mol% for mass isotopomer abundances 0.5 mol% with linear scaling up to 1 mol% for mass isotopomer abundances 25 mol%. After flux estimation, reasonably good fittings were obtained for all metabolites in each culture condition, but the computed weighted sum of squared residuals (SSR) were grossly above the expected 95% confidence interval. In an attempt to further improve fitting of the data, compartmentalization of different metabolites involved in shuttle systems between the cytosol and mitochondria (such as Mal, OAA and Cit; Ahn and Antoniewicz, 2011; 2013), and use of dilution pools previously described (such as Suc; Metallo et al., 2012) were explored. However, these modifications did not materially reduce SSR values. We then concluded the reason for the high SSR values was an inappropriate account of measurement errors, indicating that the assumed errors did not represent true experimental variability. In order to better harness this variability, we subtracted MID

5 fittings from MID measurements for all timepoints. The resulting values were then subject to a minimum cutoff of 0.3 mol% and were used as new error estimates replacing the initial default errors assumed by INCA. After this procedure, we were able to obtain flux estimation solutions with statistically accepted fits for both growth (810.4; ; 95% conf., 866 degrees of freedom) and infected cultures (853.6; ; 95% conf., 860 degrees of freedom). Ahn WS, Antoniewicz MR Metabolic flux analysis of CHO cells at growth and nongrowth phases using isotopic tracers and mass spectrometry. Metab Eng 13: Ahn WS, Antoniewicz MR Parallel labeling experiments with [1,2-13 C]glucose and [U- 13 C]glutamine provide new insights into CHO cell metabolism. Metab Eng 15: Metallo CM, Gameiro PA, Bell EL, Mattaini KR, Yang J, Hiller K, Jewell CM, Johnson ZR, Irvine DJ, Guarente L, Kelleher JK, Heiden MGV, Iliopoulos O, Stephanopoulos G Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia. Nature 481:

6 Figure S1 Experimental and simulated intracellular 13 C-labelling dynamics during CAV2 infection from [1,2-13 C]glucose. Circle markers correspond to GC-MS measurements corrected for natural isotope abundance. Lines correspond to fitted MIDs from nonstationary 13 C-MFA flux estimation of parallel labelling experiments.

7 Figure S2 Experimental and simulated intracellular 13 C-labelling dynamics during CAV2 infection from [U- 13 C]glutamine. Circle markers correspond to GC-MS measurements corrected for natural isotope abundance. Lines correspond to fitted MIDs from nonstationary 13 C-MFA flux estimation of parallel labelling experiments.

8 Table S1 - Measured metabolic uptake and production rates for MDCK cells (mock-infected) and CAV2-infected MDCK cells. Growth SD Infection SD Glc Lac NH Gln Ala Ser Glu Gly Asp Asn Arg His Thr Pro Tyr Val Met Ile Leu Lys Phe µ Specifc extracellular rates were determined during the first 24 h after label administration, in units of nmol/10 6 cells/h, except biomass formation (µ = h -1 ). Negative values indicate cellular uptake.

9 Table S2 - Measured mass isotopomer distributions and calculated errors from [1,2-13 C]glucose under growth conditions (mock infection). Time (h) PG_459 (M0) PG_460 (M1) PG_461 (M2) PG_462 (M3) PEP_369 (M0) PEP_370 (M1) PEP_371 (M2) PEP_372 (M3) Lac_261 (M0) Lac_262 (M1) Lac_263 (M2) Lac_264 (M3) Lac23_233 (M0) Lac23_234 (M1) Lac23_235 (M2) Ala_260 (M0) Ala_261 (M1) Ala_262 (M2) Ala_263 (M3) Ala23_232 (M0) Ala23_233 (M1) Ala23_234 (M2) Cit_591 (M0) Cit_592 (M1) Cit_593 (M2)

10 Cit_594 (M3) Cit_595 (M4) Cit_596 (M5) Cit_597 (M6) AKG_346 (M0) AKG_347 (M1) AKG_348 (M2) AKG_349 (M3) AKG_350 (M4) AKG_351 (M5) Suc_289 (M0) Suc_290 (M1) Suc_291 (M2) Suc_292 (M3) Suc_293 (M4) Fum_287 (M0) Fum_288 (M1) Fum_289 (M2) Fum_290 (M3) Fum_291 (M4) Mal_419 (M0) Mal_420 (M1) Mal_421 (M2) Mal_422 (M3) Mal_423 (M4) Asp_418 (M0) Asp_419 (M1) Asp_420 (M2)

11 Asp_421 (M3) Asp_422 (M4) Glu_432 (M0) Glu_433 (M1) Glu_434 (M2) Glu_435 (M3) Glu_436 (M4) Glu_437 (M5) Gln_431 (M0) Gln_432 (M1) Gln_433 (M2) Gln_434 (M3) Gln_435 (M4) Gln_436 (M5) Ser_390 (M0) Ser_391 (M1) Ser_392 (M2) Ser_393 (M3) Gly_246 (M0) Gly_247 (M1) Gly_248 (M2) Mass isotopomer distributions were corrected for natural isotope abundance. Errors were calculated as described in the supplementary text.

12 Table S3 - Measured mass isotopomer distributions and calculated errors from [U- 13 C]glutamine under growth conditions (mock infection). Time (h) PG_459 (M0) PG_460 (M1) PG_461 (M2) PG_462 (M3) PEP_369 (M0) PEP_370 (M1) PEP_371 (M2) PEP_372 (M3) Lac_261 (M0) Lac_262 (M1) Lac_263 (M2) Lac_264 (M3) Lac23_233 (M0) Lac23_234 (M1) Lac23_235 (M2) Ala_260 (M0) Ala_261 (M1) Ala_262 (M2) Ala_263 (M3) Ala23_232 (M0) Ala23_233 (M1) Ala23_234 (M2) Cit_591 (M0) Cit_592 (M1) Cit_593 (M2)

13 Cit_594 (M3) Cit_595 (M4) Cit_596 (M5) Cit_597 (M6) AKG_346 (M0) AKG_347 (M1) AKG_348 (M2) AKG_349 (M3) AKG_350 (M4) AKG_351 (M5) Suc_289 (M0) Suc_290 (M1) Suc_291 (M2) Suc_292 (M3) Suc_293 (M4) Fum_287 (M0) Fum_288 (M1) Fum_289 (M2) Fum_290 (M3) Fum_291 (M4) Mal_419 (M0) Mal_420 (M1) Mal_421 (M2) Mal_422 (M3) Mal_423 (M4) Asp_418 (M0) Asp_419 (M1) Asp_420 (M2)

14 Asp_421 (M3) Asp_422 (M4) Glu_432 (M0) Glu_433 (M1) Glu_434 (M2) Glu_435 (M3) Glu_436 (M4) Glu_437 (M5) Gln_431 (M0) Gln_432 (M1) Gln_433 (M2) Gln_434 (M3) Gln_435 (M4) Gln_436 (M5) Ser_390 (M0) Ser_391 (M1) Ser_392 (M2) Ser_393 (M3) Gly_246 (M0) Gly_247 (M1) Gly_248 (M2) Mass isotopomer distributions were corrected for natural isotope abundance. Errors were calculated as described in the supplementary text.

15 Table S4 - Measured mass isotopomer distributions and calculated errors from [1,2-13 C]glucose during CAV2 infection. Time (h) PG_459 (M0) PG_460 (M1) PG_461 (M2) PG_462 (M3) PEP_369 (M0) PEP_370 (M1) PEP_371 (M2) PEP_372 (M3) Lac_261 (M0) Lac_262 (M1) Lac_263 (M2) Lac_264 (M3) Lac23_233 (M0) Lac23_234 (M1) Lac23_235 (M2) Ala_260 (M0) Ala_261 (M1) Ala_262 (M2) Ala_263 (M3) Ala23_232 (M0) Ala23_233 (M1) Ala23_234 (M2) Cit_591 (M0) Cit_592 (M1) Cit_593 (M2)

16 Cit_594 (M3) Cit_595 (M4) Cit_596 (M5) Cit_597 (M6) AKG_346 (M0) AKG_347 (M1) AKG_348 (M2) AKG_349 (M3) AKG_350 (M4) AKG_351 (M5) Suc_289 (M0) Suc_290 (M1) Suc_291 (M2) Suc_292 (M3) Suc_293 (M4) Fum_287 (M0) Fum_288 (M1) Fum_289 (M2) Fum_290 (M3) Fum_291 (M4) Mal_419 (M0) Mal_420 (M1) Mal_421 (M2) Mal_422 (M3) Mal_423 (M4) Asp_418 (M0) Asp_419 (M1) Asp_420 (M2)

17 Asp_421 (M3) Asp_422 (M4) Glu_432 (M0) Glu_433 (M1) Glu_434 (M2) Glu_435 (M3) Glu_436 (M4) Glu_437 (M5) Gln_431 (M0) Gln_432 (M1) Gln_433 (M2) Gln_434 (M3) Gln_435 (M4) Gln_436 (M5) Ser_390 (M0) Ser_391 (M1) Ser_392 (M2) Ser_393 (M3) Gly_246 (M0) Gly_247 (M1) Gly_248 (M2) Mass isotopomer distributions were corrected for natural isotope abundance. Errors were calculated as described in the supplementary text.

18 Table S5 - Measured mass isotopomer distributions and calculated errors from [U- 13 C]glutamine during CAV2 infection. Time (h) PG_459 (M0) PG_460 (M1) PG_461 (M2) PG_462 (M3) PEP_369 (M0) PEP_370 (M1) PEP_371 (M2) PEP_372 (M3) Lac_261 (M0) Lac_262 (M1) Lac_263 (M2) Lac_264 (M3) Lac23_233 (M0) Lac23_234 (M1) Lac23_235 (M2) Ala_260 (M0) Ala_261 (M1) Ala_262 (M2) Ala_263 (M3) Ala23_232 (M0) Ala23_233 (M1) Ala23_234 (M2) Cit_591 (M0) Cit_592 (M1) Cit_593 (M2)

19 Cit_594 (M3) Cit_595 (M4) Cit_596 (M5) Cit_597 (M6) AKG_346 (M0) AKG_347 (M1) AKG_348 (M2) AKG_349 (M3) AKG_350 (M4) AKG_351 (M5) Suc_289 (M0) Suc_290 (M1) Suc_291 (M2) Suc_292 (M3) Suc_293 (M4) Fum_287 (M0) Fum_288 (M1) Fum_289 (M2) Fum_290 (M3) Fum_291 (M4) Mal_419 (M0) Mal_420 (M1) Mal_421 (M2) Mal_422 (M3) Mal_423 (M4) Asp_418 (M0) Asp_419 (M1) Asp_420 (M2)

20 Asp_421 (M3) Asp_422 (M4) Glu_432 (M0) Glu_433 (M1) Glu_434 (M2) Glu_435 (M3) Glu_436 (M4) Glu_437 (M5) Gln_431 (M0) Gln_432 (M1) Gln_433 (M2) Gln_434 (M3) Gln_435 (M4) Gln_436 (M5) Ser_390 (M0) Ser_391 (M1) Ser_392 (M2) Ser_393 (M3) Gly_246 (M0) Gly_247 (M1) Gly_248 (M2) Mass isotopomer distributions were corrected for natural isotope abundance. Errors were calculated as described in the supplementary text.

21 Table S6 - Estimated fluxes and 95% confidence intervals from combined [1,2-13 C]glucose and [U- 13 C]glutamine experiments. Growth (mock infection) CAV2 infection Reaction Flux LB UB Flux LB UB G6P <-> F6P R1 net G6P <-> F6P exch Inf Inf F6P -> FBP R FBP <-> DHAP + GAP R3 net FBP <-> DHAP + GAP exch Inf Inf DHAP <-> GAP R4 net DHAP <-> GAP exch. 5.18E+05 0 Inf Inf GAP <-> 3PG R5 net GAP <-> 3PG exch. 1.42E+04 0 Inf Inf 3PG <-> PEP R6 net PG <-> PEP exch Inf Inf PEP -> Pyr.c R G6P -> P5P + CO2 R P5P + P5P <-> GAP + S7P R9 net P5P + P5P <-> GAP + S7P exch S7P + GAP <-> E4P + F6P R10 net S7P + GAP <-> E4P + F6P exch E4P + P5P <-> GAP + F6P R11 net E4P + P5P <-> GAP + F6P exch Inf 1.53E Inf Pyr.c <-> Lac R12 net Pyr.c <-> Lac exch Pyr.c <-> Ala R13 net Pyr.c <-> Ala exch Pyr.m -> AcCoA.m + CO2 R Pyr.m + CO2 -> OAA R OAA + AcCoA.m -> Cit R Cit <-> AKG + CO2 R17 net Cit <-> AKG + CO2 exch AKG -> SucCoA + CO2 R SucCoA <-> Suc R19 net SucCoA <-> Suc exch Inf Inf Suc <-> Fum R20 net Suc <-> Fum exch Fum <-> Mal R21 net Fum <-> Mal exch. 8.80E Inf 4.52E Inf Mal <-> OAA R22 net Mal <-> OAA exch Inf Inf Mal -> Pyr.m + CO2 R

22 Cit -> OAA + AcCoA.c R Gln -> Glu R AKG <-> Glu R26 net AKG <-> Glu exch. 1.43E Inf NaN Asn <-> Asp R27 net Asn <-> Asp exch Asp <-> OAA R28 net Asp <-> OAA exch PG -> Ser R Ser -> Pyr.c R Ser <-> Gly + C1 R31 net Ser <-> Gly + C1 exch Glu <-> Pro R32 net Glu <-> Pro exch Val breakdown R NaN 8.92 Ile breakdown R Leu breakdown R Thr breakdown R Phe -> Tyr R Tyr breakdown R Met breakdown R Lys breakdown R NaN 3.38 His -> Glu + C1 R Arg -> Glu + Urea.snk R Glu + CO2 -> Arg R Pyr.c <-> Pyr.m R44 net Pyr.c <-> Pyr.m exch CO2 <-> CO2.ext R45 net CO2 <-> CO2.ext exch Inf Inf Glc.ext -> G6P R Lac <-> Lac.ext R47 net Lac <-> Lac.ext exch Ala <-> Ala.ext R48 net Ala <-> Ala.ext exch Gln.ext -> Gln R Glu <-> Glu.ext R50 net Glu <-> Glu.ext exch Asp <-> Asp.ext R51 net Asp <-> Asp.ext exch Asn <-> Asn.ext R52 net Asn <-> Asn.ext exch Ser.ext <-> Ser R53 net Ser.ext <-> Ser exch Gly <-> Gly.ext R54 net