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Amitraz PoisAmalgamation of Oncolytic Viruses with CART-cell Therapy to Target Metastatic Colorectal Cancer Stem Cellsoning Management | Case Study

Amitraz Poisoning Management | Case Study

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Amalgamation of oncolytic viruses with CART-cell therapy to target metastatic colorectal cancer stem cells





Metastatic Colorectal Cancer (mCRC) is a major cause of concern linked with higher morbidity and mortality rate worldwide. Irrespective of the recent advances in therapeutic regimen of mCRC, the overall patient survival rate remains dismal. Thus, the need of the hour is to develop enhanced treatment options focusing on personalized therapy. Therefore, the aim of the study is to establish immunomodulating and onco-suppressing targets to trigger colorectal carcinogenesis via Oncolytic Viruses (OVs) in combination with CART (Chimeric Antigen Receptor T-Cell Therapy). We hypothesize that eliminating the metastatic colorectal Cancer Stem Cells with Oncolytic Virus and CART Cells eradicates metastasis while modifying the immune environment via induction of an endogenous immune response aimed against the tumor that is indispensable for treatment success. The study will be performed using two colorectal cancer cell lines, peripheral blood and tumor tissues of the same patients. CD133+ CSCs are isolated and transfected to an oncolytic adenovirus and CART cells are then supplied to enhance anti-tumor activity. Oncolytic Virus treated with CART cells will induce apoptosis in CSC population, induce cell cycle arrest at G2/M phase, will suppress cancer cell migration and invasion, limits growth and survival of cancer cells. This combination of virotherapy and immunotherapy provides a new strategy to selectively target CSCs building a better curative option.



Key words: Cancer stem cells, Oncolytic Viruses, Metastasis, CART



Metastatic Colorectal Cancer (mCRC) is considered as one of the most prevalent malignancies maintaining the root cause for morbidity and mortality, affecting more than one million men and women and causes half a million deaths worldwide. It is the 2nd most commonly diagnosed and  2nd prominent cause of death in Canada indicating its extremely aggressive tumor behaviour (1). CRC harbors remarkable heterogeneity characterized by accumulation of several molecular alterations, genetic/epigenetic mutations and tumor microenvironment. It is a malignant epithelial neoplasm originating from the colonic or rectal mucosa, considered as adenoma growth governing uncontrolled cell growth, proliferation and tumor progression (2). Irrespective of the recent advances in therapeutic regimen of mCRC, the overall 5-year patient survival rate remains dismal. The disease continues to portend poor prognosis and the risk to develop metastasis and recurrence is higher than 50% (1-2). Distant metastasis and locoregional aggressiveness remains a major hurdle leading to failure of the present conventional chemotherapeutic modalities, signifying an urgent need to develop newer and more effective anti-cancer therapies.

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The resistance to current modalities of treatment such as chemotherapy and radiotherapy is owed to the CSC subpopulation’s ability to orchestrate recurrence and facilitate metastasis, which has significant treatment implications. Cancer stem cells has received substantial attention in recent years owing to their impending role in resistance to therapy, establishment of metastasis and recurrence (3). Cancer Stem cells, considered as the “drivers” of carcinogenesis, are a small population of self-sustaining cancer cells having high tumorigenic potential and an exclusive ability to differentiate into the heterogeneous lineages of cells that constitute the malignant tumor. Recent evidences have demonstrated significant correlations between occurrence of CSCs and disease progression, poor prognosis and disease free survival in various cancers (3). The identification and isolation of CSCs are becoming increasingly important not only for experimental cancer stem cell research but also for the development of targeted therapies. Hence, emphasizing on CSC as potential therapeutic medium for CRC, may modernize the idea of using diverse curative options for the improvisation and enhancement of the existing diagnostic and therapeutic modalities.

Furthermore, recent evidences demonstrated that off the innumerable treatment options, neither of them is sufficient in eliminating tumors nor in enduring anti-tumor immune response. Oncolytic immunotherapy, also known as virotherapy is a novel therapeutic platform that activate multimodal responses against tumours; from direct cytotoxic effects on cancer cells, to tumor associated blood vessel disruption, and eventually potent stimulation of anti-tumour immune activation (4-6). An Oncolytic Virus (OV) is defined as a genetically engineered or naturally occurring virus that can selectively and specifically target cancer cells without damaging healthy tissues. OVs are a promising class of potential next generation anti-cancer therapies that are distinguished from other therapies in a variety of features (i) OVs replicate in a tumor selective manner and can target multiple oncogenic pathways (ii) employs multiple means of cytotoxicity (iii) non-pathogenic/minimally pathogenic having least amount of side effects (iv) reverse immunosuppressive tumor microenvironment (v) resistance to OV therapy has not been seen yet (vi) in contrast with the classical drug pharmacokinetics in which the drug dosage decreases with time, in virotherapy the dose in cancer cells increases with time due to in-situ virus amplification (vii) triggers anticancer immunity (viii) sensitization to chemo-radio therapy (6-9). The tumor cell surface display distinct repertoire of receptors that facilitate tight binding which leads to successful replication of OVs. OVs have potential to synergize with CAR-T (T-cell expressing Chimeric antigen receptors) cells and can revert immunosuppression via facilitating CAR-T cell trafficking, proliferation and persistence in the tumor microenvironment (14-15) . The CART Therapy involves collecting patients own T-Cells, genetically engineered to express chimeric antigen receptors that allow T-cells to recognise and bind to a specific protein/antigen exhibited on the tumor cell surface. Once the T-cells have been manipulated, they are reproduced in the laboratory and returned to the patient. This boost of targeted immune cells is expected to result in a quick anti-cancer immune response (16-17).

The therapeutic limitations of conventional chemotherapeutic drugs present a challenge for cancer therapy; these shortcomings are largely associated to the ability of cancer cells to repopulate and metastasize after initial therapies. Joining forces with both the above mentioned field of cancer research to trigger immunotherapy that selectively targets Cancer Stem Cells for prolonged anti-tumor effects and complete remission of the disease would be of a great achievement. This imparts an impregnable rationale to establish CSC-targeted treatments to be administered in combination with immunotherapies. Therefore, in this study we hypothesize that eliminating the metastatic colorectal Cancer Stem Cells with Oncolytic Virus and CART Cells eradicates metastasis while modifying the immune environment via induction of an endogenous immune response aimed against the tumor that is indispensable for treatment success.


CAR-T cells fight metastatic colorectal cancer and enhance anti-tumor immunity

The endogenous immune system can induce immunosuppression in the tumor microenvironment and compete with adoptively transferred T cells. In this assay, CAR-T cells will improve survival of mice and enhance the efficacy of adoptively transferred T cells by eliminating immunosuppressive cells and reducing competition for homeostatic cytokines. To check the effect of CART therapy on metastasis, in the control subject, mice will be injected with mCRC CSCs and then will wait to develop lung metastasis. Another group of mice will be treated with OVs combining CART therapy. Lungs will be collected 18 days after cancer cell inoculation which will contain tumor nodules, confirming that control mice succumbed to lung metastases. CAR-T cell treatment will eliminate macroscopic tumors. CAR-T cells will also produce persistent protection against mCRC CSCs.


Oncolytic Virus treated with CART cells will induce apoptotic rate in CD133+ populationThe apoptotic rate of treated CD133+ cells will be evaluated by MuseTM flow cytometry analysis. CD133+ cells will underwent apoptosis at 48h after being transfected with OV and treated with CART cells. Cells will demonstrate a significantly increased apoptotic rate in CD133+ subpopulation compared to the control. Hence, these findings substantiate that OVs in combination with CART therapy might have a plausible role in targeting the CD133+ cancer stem cell subpopulation.

Oncolytic Virus treated with CART cells will induce cycle arrest at G2/M phase TransfectedCD133+ population will be exposed to CART cells for 48 hours to explore the possible mechanism of enhanced anti-proliferative activity. These treated CD133+ cells will demonstrate a significant decrease in the G0/G1 phase whereas a simultaneous increase will be observed in S and G2/M phases of the cell cycle. These findings will suggest that Treatment of CART on Transfected cells push CSCs from G0/G1 phase towards proliferation phase and eventually inducing cell cycle arrest at G2/M phase. Collectively, these results will prompt towards an inevitable role of combining immunotherapy in releasing the CD133 cells from G0/G1 phase thereby reducing their self-renewal potential and exerting a G2/M arrest which might responsible for inducing apoptosis in these chemoresistant cell population.


Oncolytic Virus treated with CART cells will suppress cancer cell migration and invasion

As migration and invasion is a fundamental property of CSCs random cell motility will be measured. To evaluate effects on directional cell migration, a scratch wound assay will be performed. In this assay, cells treated with vehicle were capable of completely covering the

wound area within 24 hours, whereas treated cells will reduce wound closure. Similar results will be observed for invasion assays wherein the wound area will be overlayed with Matrigel. Quantification of these results will show a dose dependent suppression of directional migration and invasion of mCRC cells. These results will highlight the efficacy of OVs to limit the motile and invasive properties of CD133+ cancer cells.

Oncolytic Virus treated with CART cells will limit growth and survival of mCRC cells

The viability of OV treated cells will be measured via a propidium iodide (PI) exclusion/uptake assay, in which epiflourescence and phase contrast images were acquired every 2 hours for a period of 48 hours. Relative to vehicle control treated cells will have increased PI-positive cells as well as reduced confluence suggesting that metastatic cancer cell growth and survival are majorly depends on the cancer stem cell subpopulation.

Significance of CD133+ cells on activation of PI3K/MAPK dual signalling pathways

To assess the probable role of mCRC CSCs in modulating the interplay between PI3K/AKT and MAPK signaling pathway, dual activation of AKT and ERK1/2 phosphorylation will be estimated in CD133+ cells using Muse Cell Analyzer. A significant decrease in dual activation of AKT and ERK1/2 phosphorylation will be observed in treated CD133+ population compared to control. The control population might demonstrate alternatively activated the MAPK pathway. These findings will be suggestive of the fact that OV mediated silencing of CD133+ stem like cells significantly inhibits the dual signalling pathway thereby alternatively pushing the cells towards the activation of an alternative pathway to maintain its proliferation and survival.

Expression profile of different genes in CD133+ subpopulation and their role in Epithelial-Mesenchymal-Transition

The effect of treated CD133+ cells on different EMT genes which are upregulated/downregulated frequently in metastatic colorectal cancers (OCT4, Nanog, SOX2, CDKN2A and E-Cadherin) and its expression profile analysis will be examined by Real-Time PCR at 24, 48 and 72-hour time intervals. Intriguingly, treated CD133+ cells will demonstrate a significant decrease in the mRNA expression patterns of OCT4, Nanog, SOX2 and CDKN2A and a notable increase in the expression levels of E-Cadherin. Thus signifying the plausible role of OVs partnered with CART in modulating the process of EMT, regulating crucial signalling pathways and altering the cell cycle mechanism even at the transcript level.

Conclusion and Future perspectives


Oncolytic virus immunotherapy is a budding and promising approach and present a new class of drugs for treating patients with cancer.  The present findings provide new insights towards selective targeting of Cancer Stem Cells CD133+ population by OVs and CARTs, strengthening their role as promising anti-cancer therapeutic agents. Research should be done to improve OV potency, efficacy and targeting. These new therapies should increase the efficacy of existing drugs against aggressive and metastatic cancers, and thus should inhibit tumor relapse and enhance prognosis. Further, these novel treatment strategies might show enhanced efficacy in synergism with the conventional chemotherapeutic agents or anti-PI3K/Wnt/KRAS inhibitors; however advance studies at tissue and circulation level are needed to substantiate this hypothesis. There are no reports displaying therapeutic targeting of CSCs in CRCs via the combination of these two immunotherapies. Most of the recently developed therapies were only cell-line based study and were tested only in in-vitro/in-vivo. Their effective doses and toxic effects in humans is still needs to be elucidated. There is a pressing need to expand the existing techniques to precisely isolate, identify and selectively target CSCs and to develop a more potent genetically engineered virus. Above all, the development of personalized combinational therapies may serve as a key to successful treatments. Furthermore, it is important to realize that the combination of chemotherapeutic drugs and Immunotherapy may have a great potential in personalized cancer therapy.



Colorectal Cancer Cell Lines, Tissues and Blood Acquirement

The human colorectal cancer cell line HCT116 and SW480 -American Type Culture Collection will be used in the study. All cell lines will be maintained in RPMI-1640 Medium (Gibco-Invitrogen, Cergy-Pontoise, France) supplemented with 10% (v/v) heat-inactivated fetal

calf serum (PAA Laboratories, Les Mureaux, France), 2mM L-glutamine, 100U/mL penicillin, and 100 g/mL streptomycin. Human colorectal primary and metastatic tumor samples from the patients and the matched peripheral blood samples will be obtained from the Kingston General Hospital. Cells will be cultured at 37C in a humidified, 5% CO2 atmosphere and will be routinely checked for  Mycoplasma contamination by PCR.

Adenovirus Construction and Identification

Oncolytic adenovirus will be packed and amplified in HCT116 and SW480 cells and as well as the patient acquired samples followed by gradient centrifugation. Virus titer will be measured by Quick Titer Adenovirus Titer Immunoassay Kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s instructions. Adenovirus genomic DNA will be extracted and applied for PCR for the following genes- OCT4, NANOG, SOX2, CDKN2A, and E-Cadherin.

Generation of CART Cells

T cells will be stimulated, expanded and transduced. Briefly, CD4+ and CD8+  T cells will be cultured separately with CD3/CD28-activating Dynabeads (Invitrogen) at a bead-to-cell ratio of  3. Approximately 24 hours after activation, T cells were transduced with lentiviral vectors. For CD8+ T cells, human IL2 (Prometheus) will be added. T cells will be counted and fed every day.

Cell Culture and Immunomagnetic Cell sorting

CRC tissues will be homogenised and mononuclear cells will be isolated from peripheral blood of mCRC patients by density gradient centrifugation. These cells will be exposed to FITC-conjugated anti-CD133 mouse antibody (Stem Cell Technologies) to identify the CD133+ cells from the heterogeneous population while the remaining cells apart from the positive subpopulation was considered as the CD133- subpopulation. Further, EasySep® FITC positive selection kit (Stem Cell Technologies) will be used to identify the FITC labelled CD133 positive cells.




Flow Cytometry analysis

TreatedCD133+ cells will be  analyzed using the MuseTM Count and Viability kit, MuseTM Cell Cycle Assay kit and MuseTMPI3K/MAPK Dual Activation kit (MuseTM Cell Analyzer; Millipore, Billerica, MA, USA) according to the manufacturer’s instructions in order to decipher the difference in their functional characteristics post transfection.

Cytotoxicity Assay

Cytotoxic killing of target cells will be measured via uptake of propidium iodide (PI) from supplemented growth medium using an IncuCyte ZOOM Live-Cell Analysis System (Essen BioScience). Transfected Cells (2 x104) were seeded in triplicate in a 96-well plate, and media with 1 μM PI (Biotium). T-cells were added and monitored every 20minutes for 3 days.

Random Cell Motility Tracking

Transfected Cells treated with T-cells will be seeded in triplicate in a 96-well plate, and placed in the IncuCyte ZOOM system.

In-vivo-Xenograft models

All in-vivo experiments will be performed according to approved protocols from the animal care committee 1×106 SW620 cells will be subcutaneously injected into the right flank of each nude mouse. When tumors reached 80‐ 120 mm3, oncolytic adenoviruses or PBS will be injected into tumors every other day for twice. The tumor volume will be measured every other day and calculated. At the end of the experiment, tumors will be resected from the sacrificed mice for further studies.

Bioluminescent Imaging

T cells expressing CBR luciferase will be used to detect trafficking of the T cells to the tumor. Anesthetized mice were imaged using a IVIS IncuCyte ZOOM system.

Quantitative Gene expression of pertinent CSC markers

Total RNA will be extracted from CD133+ treated subpopulation of colorectal cancer cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. cDNA was synthesized from 1 µg RNA of each sample using High Capacity cDNA Reverse transcription kits (Applied Biosystems). Furthermore, real-time PCR reaction mix will be  prepared in a total volume of 20 µl comprising of 2X Kapa SYBR Fast qPCR Mastermix Universal (Kappa Biosystems), 200 nM of each primer, 2.5 mM of MgCl2 and 1µl of cDNA. β-actin gene expression will be measured as endogenous control. The amplification run will be performed on the AriaMx Real-time PCR System (Agilent technologies) and the PCR reactions will be  subjected to 95ºC for 3 min followed by 40 cycles of denaturation at 95 ºC for 3 sec and annealing at 60 ºC for 20 sec, for 40 cycles. The experiment will be performed in triplicates and the relative mRNA levels were analyzed using the ddCt method after normalization with β-actin values.

Statistical Analysis 

Data will be presented as mean ± standard deviation (S.D). Mean values will be compared using an unpaired Student’s two‐tailed t‐tests. p < 0.05 will be defined as statistically significant, and p < 0.01 will be defined as extremely statistically significant.



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1)     Figure 1

CART Cells as a carrier for Oncolytic Viruses

(Chaurasiya et al, Biomedicines, 2017)

2)     Figure 2



The induction of local and systemic anti-tumour immunity by oncolytic viruses

                                             (Kaufman et al, 2004 Nature Reviews)




3)     Figure 3



       Schematic representation of OVs‐mediated brain tumor cancer stem cell targeting

(Bahreyani et al, 2018,  J Cell Biochem)

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