FIGURE 1




A)                                                                        B)                       C)
                                          500
          500                 Da13                            Rc5II
                                          400                                               0.20
 Counts




                                                                                                                                       10 % high




                                                                                                   Clonogenic capacity (%)
          400
                                          300                                                                                100
          300                                                                                                                          10 % low
                                          200
          200                                                                               0.15




                                                                          mRNA expression
                                                                                                                             80
          100                             100

            0                               0                                                                                60
                101 102 103   104   105         101 102 103   104   105                     0.10
          400
                              Co147                           Co01                                                            40
                                                                                                                                   *
                                          300
          300
 Counts




                                                                                            0.05
                                          200                                                                                20
          200

          100                             100                                                 0                               0

           0                               0
                101 102 103   104   105         101 102 103   104   105
                    CXCR4                           CXCR4
A)                                                                  B)                                            *                      FIGURE 2
                   N      T22           DITOX        H6       C
                                                                                                             *




                                                                         Internalization (a.u)
                               Linker

                   N      T22            PE24        H6       C

                               Linker                  KDEL

               T22: CXCR4 ligand and architectonic tag.
               Toxin domain: Cytotoxic protein.
               Histidine tag (H6): Architectonic tag.

                                  T22-DITOX-H6     T22-PE24-H6                                             CXCR4+ Da13    CXCR4- Co01
                   MW (kDa)             46.8           29.2
                                                                                                       T22-PE24-H6*
                   Size (nm)        38.45 ± 4.4    60.17 ± 5.03
                                                                                                       T22-PE24-H6* + AMD3100
                   Zp (mV)         -10.27 ± 0.25   -11.25 ± 0.75                                       T22-DITOX-H6*
                                                                                                       T22-DITOX-H6* + AMD3100

C)                                                   T22-PE24-H6*
                                                                    D)
                       T22-DITOX-H6*
     CXCR4+ Da13




                                                                                                 T22-DITOX-H6*            T22-PE24-H6*
     CXCR4- Co01
        A)                                                          B)                                                          FIGURE 3
                                                                                       *     *
                                T22-PE24-H6 IC50: 0.15 µM
                                T22-DITOX-H6 IC50: 0.06 µM




                                                                 Cell viability (%)
  Cell viability (%)
                                                                                                                T22-PE24-H6
                                                                                                                T22-PE24-H6 + AMD3100
                                                                                                                T22-DITOX-H6
                                                                                                                T22-DITOX-H6 + AMD3100




                                                                                      CXCR4+ Da13 CXCR4- Co01

                             Concentration (µM)
      C)                                                            D)


                                                                                                                      5-FU (1 µM)
                                                             Cell survival (a.u)                                      Untreated
                                                                                                                      Oxaliplatin (1 µM)
Cell viability (%)




                                                                                                                 T22-DITOX-H6 (0.1 µM)
                                     Counts




                                                                                                                T22-DITOX-H6 (1 µM)
                                                 CXCR4

                       WT Colon organoids
                                                                                             Time (Days)
A)                                                B)                                                                       FIGURE 4
       Da13 CXCR4+           Co01 CXCR4-                                    50         Da13 CXCR4+
                                                                                                                     *




                                                       Single cell deaths
                                                                                       Co01 CXCR4-
                                                                            40


                                                                            30                                 *
                                                                            20
                                                                                                     *
                                                                            10


                                                                             0
                                    CXCR4 1:100                                   control        5h            24h   48h

C)                 Control        5h                                        24h                          48h
     Da13 CXCR4+
     Co01 CXCR4-
A)                                                                                          FIGURE 5
              Control   5h   24h   48h
Da13 CXCR4+




                                                             20                 µ,*
                                                             18
                                                                  µ,α,β




                                         Mitotic figures
                                                             16
                                                             14
                                                             12                           *, α
                                                             10                                  *, β
                                                              8
                                                              6
Co01 CXCR4-




                                                              4
                                                              2
                                                              0
                                                                  control        5h        24h   48h

                                                                  Da13 CXCR4+
                                                                  Co01 CXCR4-
                                                                                            α
B)                                                                          *         •
              Control   5h   24h   48h




                                         % Ki67 expression
Da13 CXCR4+




                                                                  control       5h         24h   48h
                                                                                                        FIGURE 6 AB


A)          Control +    Control (K)       5h              24h                 48h
                                                                                             CXCR4 (H-Score)
 CXCR4




                        H-score:46.5    H-score:45       H-score:73          H-score:94
B)          Control +     Control (K)       5h             24h                 48h
 H2Gamma




                                                                                              K    5h     24h   48h




                              8%           15%              27%                  37%
                                                                                             H2Gamma (%)
                                                                                                                *
 PARP




                                                                                             *p=0.006


                           negative       negative   5%, low intensity   7%, low intensity
 Caspase3




                                                                                              K    5h     24h 48h
                           negative       negative         negative             negative
                                                              FIGURE 6 C
C)
                  Control   5h   24h   48h
                                             Caspase 11 (H-Score) α
                                                  *p=0.007
     Caspase 11




                                                  •p=0.02
                                                  αp=0.033




                                                       *       •
                                              K       5h      24h   48h
                                             NLPR3 (H-Score)         α

                                                  *p=0.001
                                                  •p=0.008
      NLPR3




                                                  αp=0.0001
                                                               •
                                                       *

                                              K       5h      24h   48h
                                                                                                                         FIGURE 7
A)                                                                                                 C)
 Subcutaneous tumour volume (mm3)


                                    900                                                                 Kidney   Liver
                                    800       Control
                                    700       5-FU
                                    600       T22-DITOX-H6




                                                                                                                                      Control
                                    500                                                 p=0,014

                                    400
                                                                                         p=0,026
                                    300

                                    200

                                    100




                                                                                                                                    T22-DITOX-H6
                                      0
                                          1     3         5       7       9        11

                                    30              Time (days)
B)
                                    25

                                    20
 Body weigth (mg)




                                    15
                                              Control




                                                                                                                                    5-FU
                                    10        5-FU
                                              T22-DITOX-H6
                                     5

                                     0
                                          1    3      5       7       9       11
                                                    Time (days)
GRAPHICAL ABSTRACT