Cell culture and establishment of our stable primary cell lines:
1. Sample collection
Specimens from two patients (one with bladder cancer and no schistosomiasis and the other with a case of schistosoma-associated bladder cancer) were utilized in this study. The study was approved by the AFCM’s Institutional Review Board and ethics committee. The patients signed informed consent forms and two specimens were collected from each patient; a blood sample (used to isolate T cells) and a tissue biopsy (used to create a primary bladder cancer cell line) obtained during cystoscopy.
2. Separation of human lymphocytes using Ficoll-hypaque
3. Isolation of CD3/CD28 human T cells using Dynabeads
(pictures are needed for every section)
Putting the first circuit (siRNA) together:
- We performed digestion of our plasmid (pSB1C3) and parts (14 and 15) to put together our first circuit (the siRNA circuit):
Lane 2: siRNA fragments ligated Lane 3: siRNA Ligated to pSB1C3+ (10 kb Ladder lane adjusted)
Part BBa_K3244024 (siRNA cassettes for optimizing CAR-T cells for solid tumors) has been composited into part BBa_K3244025 which consists of triple cassettes for U6, TOX and NR4A each consists PD-1 promoter, sense, hairpin loop, antisense and terminator. To form vector you will need RSV promoter, U6 promoter. bGh poly(A) for termination in mammalian cells. In our case we also added AmpR and AmpR promoter to compensate for the unavailability of chloramphenicol for research purposes.
Putting the second circuit (CAR) together:
- We performed digestion of our plasmid (pcDNA) and parts (1 and 2) to put together our first circuit (the CAR circuit):
Lane2: CAR HDR Ligated to pcDNA3.1+ (Ladder lane adjusted)
Part BBa_K3244027 which represents a composite device of 2nd Generation CAR-T, part BBa_K3244028 which represents a composite IL-18 expression construct for TRUCK CAR design and finally BBa_K3244029 which represents CRISPR Homology Directed Repair template with both L-Arm and R-Arm for IL-18-producing TRUCK CAR-T Cell
Our first transfection (siRNA):
- We transfected T cells from both patients with the siRNA circuit using lipofectamine 3000.
Figure: PD-1 knocked out CD3/CD28 cultured cells (X100) | PD-1 knocked out CD3/CD28 cultured cells (X400) | PD-1 knocked out CD3/CD28 cultured cells (X1000) |
We performed gene Expression analysis of cultured T cell that was isolated from BC patients and engineered by using our first circuit by Real time quantitative PCR (qPCR) using Specific oligonucleotide sequences for NR4a and PD-1. PCR was performed on 5-plex Rotor Gene PCR instrument, Qiagen, Hilden, Germany
Serial | group | Ct (NR4a) | Ct(ACTB) | ΔCt (NR4a) | ΔΔCt(NR4a) | RQ(NR4a) |
1 | T cell from BC associated Schitosoma [silenced] | 38.82 | 36.26 | 2.56 | -3.59 | 0.08 |
2 | T cell from BC negative for Schitosomiasis [silenced] | 40.19 | 32.13 | 8.06 | 1.91 | 3.75 |
3 | T cell BC [control] | 36.29 | 30.14 | 6.15 | 0 | 1 |
Serial | group | Ct (PD-1) | Ct(ACTB) | ΔCt (PD-1) | ΔΔCt(PD-1) | RQ(PD-1) |
1 | T cell from BC associated Schitosoma [engineered] | 35.82 | 30.25 | 5.57 | 4.75 | 26.99 |
2 | T cell from BC negative for Schitosomiasis [engineered] | 32.08 | 26.75 | 5.33 | 4.51 | 22.783 |
3 | T cell BC [unengineered] | 38.11 | 37.29 | 0.82 | 0 | 1 |
Δ:Delta, Ct: Cycle threshold, RQ: quantitative gene expression relative to housekeeper gene, ACTB: ꞵ-actin
A significant downregulation of PD1 and NR4A was observed in engineered patient-derived T-cells compared to un-engineered cells (p<0.01), however, higher levels were obtained in cells derived from the Schistosoma associated BC patient, although significance was not achieved (p<0.05). On the other hand, NR4a was decreased in engineered T cells (p<0.05). The gene expression was calculated according to un-engineered T cells (negative control)
We then performed flow cytometry using a PD-1 specific monoclonal antibody:
Figure 1: flow cytometric detection of PD-1 in T cells of the schistosoma-negative bladder cancer patient post-interference using the siRNA circuit
Figure 2: flow cytometric detection of PD-1 in T cells of the schistosoma-positive bladder cancer patient post-interference using the siRNA circuit
PD-1 siRNA negative Control Schistosoma-negative BC Schistosoma-associated BC
- Our results showed that schistosoma-associated BC showed high titres of isolated T cells and a higher percentage of PD-1 knocked-down cells in the population (47.2%). In the case of schistosoma-negative BC, lower titres of T cells were initially isolated and the percentage of T-cells with successful PD-1 knockdown was significantly lower (11.4%).
Immunofluorescence staining of T-cells using PD-1 specific antibody
Image 1: Immunofluorescence staining of un-engineered T cells isolated from Peripheral blood sample of Bladder cancer patients, the cells was primary cultured in CTS optimizer media, harvested cells were stained with unconjugated monoclonal anti-PD-1 antibody (green). An AlexFluor 488 anti-rabbit IgG secondary antibody was used for detection. Un engineered cells showed a small colonies of normal sized T cells that showed homogenous dense expression of PD-1 protein with high dense fluorescence intensity (+++), fluorescence was examined by immunofluorescence microscope (Labmed; USA), the used magnification power was X400
Image 2: Immunofluorescence staining of engineered T cells isolated “Knock-out of PD-1”from Peripheral blood sample of Schistosoma associated Bladder cancer patients, the cells was primary cultured in CTS optimizer media, harvested cells were stained with unconjugated monoclonal anti-PD-1 antibody (green). An AlexFluor 488 anti-rabbit IgG secondary antibody was used for detection. Engineered cells showed a large colonies of large sized T cells that showed homogenous low expression of PD-1 protein with moderate faint fluorescence intensity (+), fluorescence was examined by immunofluorescence microscope (Labmed; USA), the used magnification power was X400.
Image 3: Immunofluorescence staining of engineered T cells isolated “Knock-out of PD-1”from Peripheral blood sample of Schistosoma negative Bladder cancer patients, the cells was primary cultured in CTS optimizer media, harvested cells were stained with unconjugated monoclonal anti-PD-1 antibody (green). An AlexFluor 488 anti-rabbit IgG secondary antibody was used for detection. Engineered cells showed a large colonies of large sized T cells that showed homogenous moderated expression of PD-1 protein with moderate bright fluorescence intensity (++), fluorescence was examined by immunofluorescence microscope (Labmed; USA), the used magnification power was X400.
Our second transfection (CAR):
Using the CRISPR protocol for editing of T cells, we introduced the second circuit into the two T cells with successful PD-1 knockdown (derived from patients with schistosoma-associated and non-schistosoma associated BC). We confirmed the stable integration of our circuit using RealTime PCR.
Gene Expression analysis of cultured T cell that was isolated from BC patients and engineered by our CAR circuit by Real time quantitative PCR (qPCR) using Specific oligonucleotide sequences for HDR1, PCR was performed on 5-plex Rotor Gene PCR instrument, Qiagen, Hilden, Germany
Δ:Delta, Ct: Cycle threshold, RQ: quantitative gene expression relative to housekeeper gene, ACTB: ꞵ-actin
Serial | group | Ct (HDR1) | Ct(ACTB) | ΔCt (HDR1) | ΔΔCt(HDR1) | RQ(HDR1) |
1 | T cell from BC associated Schitosoma [engineered] | 28.51 | 30.25 | -1.74 | -3.84 | 14.3 |
2 | T cell from BC negative for Schitosomiasis [engineered] | 30.41 | 31.07 | -0.66 | -2.76 | 6.77 |
3 | T cell BC [unengineered] | 38.26 | 36.15 | 2.11 | 0.01 | 0.99 |
Co-culturing our engineered T cells with BC primary cell lines:
- We co-cultured our engineered T cells with schistosoma-associated and non-schistosoma-associated BC cells. 48 hours later we measured apoptosis using Annexin V assay
Figure: Annexin V apoptosis of Schistosomiasis-associated BC cells after co-culture with engineered T cells (FCM)
BC cells that was treated with CAR-t cells engineered for ERB2 and Schistosoma egg antigen showed a significant apoptosis (59.4%) and 12% dead tumor cells
Figure: Annexin V apoptosis of Schistosomiasis-negative BC cells after co-culture with engineered T cells (FCM)
BC cells that was treated with CAR-t cells engineered for ERB2 and Schistosoma egg antigen should a significant apoptosis (54.8%) and 21% dead tumor cells.
Comparison between cytotoxic effect of engineered T cells on both types of BC cell lines in vitro:
CAR-T treated BC cells negative for Schistosoma ( after 48 hours) | CAR-T treated BC cells associated Schistosoma ( after 48 hours) |
Cell counts of BC cell lines 48 hours after co-culture:
Variable | BC negative for Schistosomiasis "treated" | BC associated Schistosomiasis "treated" | untreated BC | |
cell count | 1 | 1.55E+05 | 1.41E+05 | 2.87E+05 |
2 | 1.67E+05 | 1.25E+05 | 2.64E+05 | |
3 | 1.75E+05 | 1.06E+05 | 2.71E+05 | |
mean of triplicate | 1.66E+05 | 1.24E+05 | 2.74E+05 | |
dead cells | 1 | 6.20E+04 | 5.20E+04 | 2.22E+03 |
2 | 5.90E+04 | 5.20E+04 | 1.10E+03 | |
3 | 6.50E+04 | 8.80E+04 | 1.90E+03 | |
mean of triplicate | 6.20E+04 | 6.40E+04 | 1.74E+03 | |
viable cells | 1.04E+05 | 6.00E+04 | 2.72E+05 | |
cell viability(%) | 62.58% | 48.39% | 99.36% |
High reduction of viable BC cells that was treated by CAR-t was demonstrated in Schistosomiasis associated BC compared to unassociated cells, but no statistical significance was achieved (p>0.05)
After co-culture, we also performed MTT assay to assess cellular proliferation:
BC negative for Schistosomiasis "treated" | BC associated Schistosomiasis "treated" | untreated cells | ||
OD at 570 nn | 1 | 0.815 | 0.654 | 1.258 |
OD at 570 nn | 2 | 0.782 | 0.626 | 1.294 |
OD at 570 nn | 3 | 0.825 | 0.545 | 1.105 |
mean of triplicate | 0.81 | 0.61 | 1.22 | |
cell viability(treated-untreated) | 0.36 | 0.55 | 1.16 | |
cell viability (%) | 69.32% | 52.16% | ||
%inhibition | 30.7 | 47.8 | ||
MTT: (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- A statistical significance difference was achieved between the % of viability in CAR-t treated BC cells associated Schistosomiasis and unassociated cells (p<0.05).
- Moreover, between untreated and CAR-t treated BC cells (p<0.01)
