Research ArticleCancer

CRISPR-enhanced engineering of therapy-sensitive cancer cells for self-targeting of primary and metastatic tumors

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Science Translational Medicine  11 Jul 2018:
Vol. 10, Issue 449, eaao3240
DOI: 10.1126/scitranslmed.aao3240
  • Fig. 1 Concept of study and identification of DRL for cancer cell–based self-targeting therapies.

    (A) Allogeneic approach: Cancer cells resistant to receptor-targeted therapies can be used off-the-shelf for delivery of receptor ligands toward allogeneic cancers with ligand-sensitive phenotypes in the setting of primary treatment. Because they are co-engineered with a prodrug-activatable suicide system [herpes simplex virus thymidine kinase (HSV-TK)], therapeutic cancer cells can be eliminated after therapy using ganciclovir (GCV). (B) Autologous approach: Cancer cells harvested from patients during initial surgery and identified as sensitive to receptor-targeted therapies can be CRISPR-engineered to knock out the target receptors. Receptor knockout (KO) results in therapy resistance and allows engineering with receptor ligands and delivery toward autologous self-tumor sites in the setting of recurrence. (C) A panel of primary and metastatic cancer cell lines was treated with conditioned medium containing secretable variants of different receptor-targeted molecules, and viability was assessed 72 hours after treatment (n = 3 technical replicates). TSP, thrombospondin-1; Nb, nanobody. (D) Cancer cell lines were tested with varying concentrations of TRAIL to quantify TRAIL sensitivity (n = 3 technical replicates). TRAIL-sensitive (s) or -resistant (r), nodular (n) and invasive (i) glioblastoma (GBM), colon cancer (CC), T cell leukemia (TCL), metastatic breast cancer (BCm), and metastatic prostate cancer cells (PCm) were tested. All in vitro experiments were repeated at least twice. Means ± SD are shown. P values by unpaired t test. **P < 0.01, ***P < 0.001.

  • Fig. 2 Off-the-shelf therapy using DRL-resistant self-targeting tumor cells.

    (A) Concept of off-the-shelf approach: Receptor ligand (RTL)–resistant tumor cells can be engineered to secrete RTL, which can induce cell death in RTL-sensitive allogeneic cancer cells. (B) Cell viability of DRL sGBMi-FmC cells during time course coculture with DRL-resistant cancer cell lines (rGBMi1 or rGBMi2) expressing secretable DRL ST (n = 3 technical replicates). (C) Caspase 3/7 activity in sGBMi-FmC 8 hours after start of coculture with ST-expressing rGBMs (n = 2 technical replicates). (D) Western blot analysis of poly(adenosine 5′-diphosphate–ribose) polymerase (PARP), caspase 8 cleavage, and α-tubulin in DRL sGBMi-FmC 8 hours after the start of coculture with either GFP or ST-expressing rGBM. (E) In vitro cell viability (n = 3 technical replicates) and (F) in vivo growth of DRL-resistant cancer cells (rGBMi2) co-engineered with prodrug-converting enzyme HSV-TK and GFP-Fluc (GFl) in the absence or presence of GCV over time (n = 5 per group). (G) In vivo positron emission tomography (PET)–based monitoring of rGBMi2 co-engineered with ST and HSV-TK (rGBMi2-ST-TK) with and without GCV treatment (n = 3 mice per group). (H) Evaluation of bystander effect of rGBMi2-ST-TK cells on cocultured sGBMi-FmC cells over time (n = 3 technical replicates). (I) sGBMi2-GFP cells (5 × 105) were implanted at a distance of 1.5 mm from established sGBMi-FmC tumors. Representative fluorescence photomicrograph shows cell populations 2 weeks after injection of sGBMi2-GFP (n = 2 mice). Scale bar, 200 μm. (J) Top: Experimental outline for testing efficacy of rGBMi2-ST-TK in mice bearing intracranial sGBMi-FmC tumors. Bottom: Estimate of relative tumor volume in treatment groups based on Fluc signal of sGBMi-FmC–bearing mice (left) and the respective Kaplan-Meier survival curves (right); n = 3 for phosphate-buffered saline (PBS), n = 5 for rGBMi2-GFP, and n = 6 for rGBMi2-ST-TK + GCV. All in vitro experiments were repeated at least twice. Means ± SD are shown for in vitro experiments and means ± SEM are shown for in vivo experiments. P values by unpaired t test (B and C) or Mantel-Cox (log-rank) test (J), *P < 0.05, ***P < 0.001.

  • Fig. 3 CRISPR-mediated DR-KO and influence on sensitivity to DRL TRAIL.

    (A) Concept of autologous approach: CRISPR-mediated receptor KO changes the phenotype of cancer cells from RTL-sensitive to RTL-resistant before engineering with secretable RTL. (B) Western blot analysis of DRL-sensitive tumor lines transduced with doxycycline-inducible FLAG-Cas9 or constitutively expressed FLAG-Cas9 (sBCm) constructs, blotted for FLAG and total extracellular signal–regulated kinase (ERK). (C) Single-clone Western blot analysis of CRISPR-targeted DR4, DR5, and total ERK expression in sGBMn. (D) Flow cytometry analysis of DR4 and DR5 surface expression in wild-type cells and KO clones identified in (C). (E) Single-clone sequencing of genomic DNA from wild-type and KO sGBMn clones identified in (C). (F) Titration of DR-KO clones with DRL TRAIL (n = 3 technical replicates). (G) Western blot analysis of PARP and caspase 8 cleavage and total ERK in DR wild type (sGBMn-FmC, sGBMn-Cas9, and sGBMnRec-FmC) and DR single- or double-KO clones identified in (C) 8 hours after treatment with DRL TRAIL. (H) Western blot analysis of DR4, DR5, and total ERK expression in other DR-KO CRISPR-engineered tumor cell lines.

  • Fig. 4 In vitro autologous self-targeting efficacy of DR-KO tumor cells co-engineered with a secretable DRL and a suicide system.

    (A) Strategy for the establishment of autologous recurrent glioblastoma models using in vivo TMZ treatment. (B) Effect of in vivo TMZ treatment on intracranial growth of nodular (sGBMnRec-FmC) and invasive (sGBMiRec-FmC) tumors as monitored by BLI (n = 1 each; see also tables S1 and S2). (C) Recurrent tumor lines established in (B) and their respective primary lines were titrated with TMZ (left) and DRL TRAIL (right) to identify differences in their sensitivity to TMZ and TRAIL treatment (n = 3 technical replicates each). (D) Representative photomicrographs (top) and assessment of viability (bottom) of DRL sGBMnRec-FmC or sGBMiRec-FmC cocultured with increasing percentages (0 to 150%, as indicated on the x axis) of their respective autologous TRAIL-secreting cell lines or autologous GFP-transduced controls (n = 3 technical replicates). Scale bars, 200 μm. (E) GCV titration of DR4/5 KO cancer lines engineered with or without prodrug-converting suicide system HSV-TK in vitro (n = 3 technical replicates). All in vitro experiments were repeated at least twice. Means ± SD are shown. P values by unpaired t test. **P < 0.01, ***P < 0.001.

  • Fig. 5 In vivo autologous self-targeting efficacy of DR-KO tumor cells co-engineered with a secretable DRL and a suicide system.

    (A) Photomicrograph time course of sECM-encapsulated ST-secreting rGBMnDR4/5-ST-TK cocultured with their autologous DR wild-type parental cells (sGBMnRec-FmC). Scale bar, 200 μm. (B) rGBMnDR4/5-ST-TK was engineered with GFl. The graph on the left shows in vitro correlation of Fluc signal with cell number. rGBMnDR4/5-ST-TK-GFl was encapsulated in sECM, followed by intracranial implantation into SCID mice. The graph on the right shows Fluc signal of rGBMnDR4/5-ST-TK-GFl before and after GCV treatment (n = 2 mice). (C) Top: Experimental outline for testing efficacy of sECM-encapsulated rGBMnDR4/5-ST-TK in mice with resected sGBMnRec-FmC tumors. Photomicrographs show light and fluorescence photos of intact and resected intracranial tumors after implantation of therapeutic cells. Scale bars, 1 mm. Black/white dashed circles indicate tumor area. The bar graph on the right shows mean tumor volume estimated on the basis of Fluc signal before and after resection (n = 28). Middle: Estimate of relative tumor volume after resection in treatment groups based on Fluc signal intensity of sGBMnRec-FmC-bearing mice (left). Kaplan-Meier survival curves are shown on the right (control, n = 4; resection alone, n = 4; rGBMnDR4/5-ST-TK -GCV, n = 11; rGBMnDR4/5-ST-TK + GCV, n = 13). Bottom: Representative hematoxylin and eosin (H&E)–stained sections and immunofluorescence photomicrographs of nonresected control versus resected sGBMnRec-FmC tumors treated with therapeutic rGBMnDR4/5-ST-TK with or without GCV. Scale bars, 200 μm. (D) Top: Experimental outline for testing the efficacy of rGBMiDR4/5 (control) or rGBMiDR4/5-ST-TK in mice bearing intracranial sGBMiRec-FmC tumors. Middle: Estimate of relative tumor volume in treatment groups based on Fluc signal of sGBMiRec-FmC–bearing mice (left) and respective Kaplan-Meier survival curves (right) (rGBMiDR4/5, n = 7; rGBMiDR4/5-ST-TK − GCV, n = 9, rGBMiDR4/5-ST-TK + GCV, n = 8). Bottom: Immunofluorescence photomicrograph of sGBMiRec-FmC–bearing mice injected with rGBMiDR4/5-ST-TK (no GCV treatment). Scale bar, 200 μm. (E) Top: Experimental outline for testing efficacy of rBCmDR4/5-ST-TK injected via the internal carotid artery (ICA) in mice bearing intracranial sBCm-RmC tumors. Bottom: Estimate of relative tumor volume increase based on Rl signal intensity of sBCm-RmC–bearing mice (left) and respective Kaplan-Meier survival curves (right) (PBS, n = 3; rBCmDR4/5-ST-TK − GCV, n = 3; rBCmDR4/5-ST-TK + CGV, n = 4). Means ± SEM are shown. P values by unpaired t test (C, top) or Mantel-Cox (log-rank) test (survival curves), *P < 0.05, **P < 0.01, ****P < 0.0001.

  • Fig. 6 Migratory potential of CRISPR-engineered therapeutic tumor cells toward recurrent self-tumor sites.

    (A) Experimental outline: 5 × 105 sGBMiRec-FmC cells were implanted into the right hemisphere of SCID mice, followed by injection of 5 × 105 rGBMiDR4/5-GFP cells at a distance of 1.5 mm laterally 3 days later. Mice were sacrificed at days 1, 7, 14, and 28 after rGBMiDR4/5-GFP implantation (n = 2 for each time point) to assess migration of CRISPR-engineered rGBMIDR4/5-GFP cells toward the sGBMiRec-FmC self-tumor site. (B) Representative fluorescence photomicrographs showing the location of sGBMiRec-FmC (red) and rGBMiDR4/5-GFP (green) tumor cell populations at the time points outlined above. The dashed line was placed adjacent to the rGBMiDR4/5-GFP implantation site to facilitate quantification of migration toward the established sGBMiRec-FmC tumor site. The red box marked in the photomicrograph for day 28 is magnified in (C). Scale bars, 200 μm. (C) Magnified fluorescence microphotograph from day 28. Scale bar, 100 μm. (D) Quantification of rGBMiDR4/5-GFP migration toward the sGBMiRec-FmC tumor site at different time points based on rGBMiDR4/5-GFP cell count from the left part of (B), excluding the nonmigratory established tumor site shown to the right of the dashed line. Means ± SD are shown. Two biological replicates per time point.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/449/eaao3240/DC1

    Materials and Methods

    Fig. S1. DR expression, engineering of DRL-resistant cancer cells with Rl-ST, and in vitro coculture efficacy.

    Fig. S2. Screening and identification of CRISPR-induced DR-KO.

    Fig. S3. Engineering of cell lines for in vivo BLI and ST expression from DR4/5 KO cell lines.

    Fig. S4. ELISA quantification of secreted TRAIL from CRISPR-engineered therapeutic cancer cell lines.

    Fig. S5. Concept of autologous cancer cell–based self-targeting strategies and possible role of GCV-activated HSV-TK suicide system in case of DRL nonresponsive tumor recurrence.

    Table S1. Establishment of sGBMnRec-FmC recurrent cell line via in vivo TMZ treatment of sGBMn-FmC (provided as an Excel file).

    Table S2. Establishment of sGBMiRec-FmC recurrent cell line via in vivo TMZ treatment of sGBMi-FmC (provided as an Excel file).

    References (7078)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. DR expression, engineering of DRL-resistant cancer cells with Rl-ST, and in vitro coculture efficacy.
    • Fig. S2. Screening and identification of CRISPR-induced DR-KO.
    • Fig. S3. Engineering of cell lines for in vivo BLI and ST expression from DR4/5 KO cell lines.
    • Fig. S4. ELISA quantification of secreted TRAIL from CRISPR-engineered therapeutic cancer cell lines.
    • Fig. S5. Concept of autologous cancer cell–based self-targeting strategies and possible role of GCV-activated HSV-TK suicide system in case of DRL nonresponsive tumor recurrence.
    • References (7078)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1. Establishment of sGBMnRec-FmC recurrent cell line via in vivo TMZ treatment of sGBMn-FmC (provided as an Excel file).
    • Table S2. Establishment of sGBMiRec-FmC recurrent cell line via in vivo TMZ treatment of sGBMi-FmC (provided as an Excel file).

    [Download Tables S1 and S2]

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