18/12/2019
Cancer Stem Cells in colorectal surgery
INTRODUCTION
Trends in the incidence and mortality curves over the last decade testify the success of early diagnosis and the effectiveness of treatments implemented in patients with Colorectal Carcinoma (CRC). Full agreement exists in acknowledging surgery as the only radical treatment amenable to be combined with radiotherapy and chemotherapy1.
Nevertheless, colorectal cancer is still the second cause of death for malignant disease in industrialized countries, after prostate cancer in men and breast tumor in women.
About half of patients who have a radical and curative resection is at risk of recurrence2. Moreover, surgery combined with chemotherapy and radiotherapy has prolonged survival of patients with metastatic CRC, but their prognosis remains poor.
In 2007 Colorectal Cancer Stem Cells (CR-CSC) were isolated and expanded through a methodology in vitro and in vivo, reproducing the patient tumor in immunocompromised mice3.
Since then many studies have highlighted that CR-CSCs are responsible for tumor propagation, metastatic dissemination, relapse and chemoresistance. However, molecular mechanisms underlying therapy resistance remain still largely unknown.
In this work, we present the results of a research, carried out in collaboration with the “Istituto Superiore di Sanità”, that was set up with the aim of isolating the Cancer Stem Cells (CSCs), which grow into colorectal tumours. We tested colorectal tissues and grew the cells in vitro in order to set up a large bio-bank. As part of this research, our attention was focused on the study of CSCs in the examined samples, and we tried to unravel the correlation between the expression of CSC and the aggressiveness of the tumour.
MATERIAL AND METHODS
Fresh human colorectal cancer tisuues were obtained in accordance with the standards of the ethics committee on human experimentation of the Istituto Superiore di Sanità (authorization n. CE5ISS 09/282).
Samples of colorectal tumour tissues were collected from 22 patients (14 males and 8 females), who underwent colorectal surgery.
All patients were operated by the same surgical team at the Department of Surgical Sciences, Policlinico Umberto I, Sapienza, University of Rome, between January 2014 and June 2017.
Tissue sample collection was carried out by dedicated personnel (surgeons and anatomo-pathologists), who isolated 1 cm3 of healthy mucosa, at a distance from the tumour site, and 1 cm3 of the tumour lesion. The samples were stored and transported in special, separate tubes. The sample from the operation was then subjected to routine definitive anatomo-pathological examination.
For each patient, the following data were included: tumour site, histotype, grading, disease staging, adjuvant therapy (where applicable) and survival.
Only patients with colorectal cancer, who had not undergone neo-adjuvant radio and/or chemotherapy and had non-ulcerated tumours greater than 1 cm in diameter were included in the series. All patients were informed about the procedure and expressed their full consent. The follow up was performed after 6 months, and then annually to evaluate the responses to chemotherapy, relapse rates and survival rates
CSC isolation and culture
Fresh human colorectal cancer tissues were processed within 24-48 hours after surgical resection. Clusters of proliferating cells became evident after a variable time length ranging from 5-7 days to 3 weeks. Cultures in which no proliferating clusters were detected after 4 weeks were discarded. Spheroids were then passaged 1:2 weekly by mechanical dissociation or by incubation.
Animal procedures
All animal procedures were performed according to the national Animal Experimentation guidelines (D.L.116/92) upon approval of the experimental protocol by the Institutional Animal Experimentation Committee. 4 to 6-week-old female NOD were used for all the validations and experiments. For CSC validation, 5x105 cells were injected subcutaneously in the flank of 3 replicate mice. Palpable xenografts were extracted and evaluated by a pathologist in comparison with the human tumor of origin.
Mice were recorded negative when no graft was observed after 24 weeks from the inoculum.
The results were validated successively by Flow cytometry, Clonogenicity assay, Reverse-Phase Protein Array (RPPA) Analysis, Western blotting and Viability assay4.
RESULTS
Molecular studies are still ongoing, and the results will be published by the “Istituto Superiore di Sanità”.
Our series consisted of 22 patients with colorectal cancer: 14 males and 8 females, with an average age of 69.77 years (range: 54-84).
Reported in Table 1 are: tumour site, histotype, grading, disease staging, adjuvant therapy (where applicable) and survival.
The mean follow-up was….months.
Tumour stem cells were isolated from 5 patients. The 5 patients, in whom the CSC were isolated, had a neoplasm at an advanced stage of disease (Stages III and IV) and with unfavourable grading (G3-G4).
3 of the 5 patients died at a distance of -, -, - months after the intervention.
The other two patients were still alive at a distance of --- and - months from the intervention. They underwent Chemotherapy and RSCS after…, which did not show any disease recurrence.
DISCUSSION
The CSC theory is based on the hierarchical organisation of the cells that make up the tumour. According to this hypothesis, the ability to start and sustain the growth of a tumour is the prerogative of a small percentage of undifferentiated cells with the characteristics of staminality, called tumour stem cells. The results largely support the validity of this hypothesis for colon carcinomas. In the latter the tumorigenic potential is confined to a sub-population of undifferentiated cells.
The sub-cutaneous inoculation of a small fraction of tumour cells in immuno-compromised mice is capable of faithfully reproducing, from both a histological and molecular perspective, the parental tumour.
Selective culture conditions allowed us to grow CSCs in vitro, in the form of cell aggregates called tumour spheres. These spheres maintain an undifferentiated phenotype. In vivo studies on mouse models have confirmed the tumorigenic potential of these spheres, which are able to reproduce the parental tumour, even after they have been growing in culture as undifferentiated cells for more than a year. These data provide further confirmation of the high proliferative potential and self-renewal of isolated cells in colon tumours.
Several models have been developed to support the emerging consensus that CSCs derive from the temporal accumulation of genetic/epigenetic mutations5,6,7. In fact, the complex transformation process can involve diverse stages, and can occur in parallel with micro-environment deregulation, associated with telomere attrition, inactivation of tumour suppressor genes and the upregulation of oncogenes, along with genomic and epigenetic instability. In the more advanced stages of malignant transformation, the inactivated CSCs can evade the immune system and acquire metastatic capabilities8.
Isolation of CSCs in these and other types of solid tumours, including glioblastoma9, melanoma10, prostate carcinomas11, breast and pancreatic carcinomas12,13 has profoundly influenced cancer research in recent years, and determined the development of new approaches that are aimed at understanding the molecular function of CSCs and consequently, the development of target therapies.
One of the aspects that needs further investigation is the identification of biomarkers that render techniques for isolating stem cell cancer cells increasingly effective and selective.
Another aspect that needs to be considered is the tumour micro-environment: in order to understand the behaviour of CSCs, we must necessarily consider: the niches for CSCs, the stroma, the immune system and tumour vascularisation14. Obviously there are a large number of details that have yet to be clarified; in the future, one might think of tumours as "organs", as they are composed of several types of tissue (with different embryonic origins)15.
CSCs are primarily responsible for tumour progression, resistance to chemotherapy, metastases and relapses.
In patients who have received what appears to be radical surgery, the risk of recurrence varies with the pathological stage of the primary tumour. The increasing onset of aggressive CCR in young adults has highlighted changes in the nature of this disease16.
The CSC theory may also explain tumour recurrence after a tumour has been completely removed by surgery (macroscopic R0), or after a seemingly complete response to chemotherapy. It is possible that these cancer stem cells can nest themselves down in "safe" sites (niches) in our body, where they can remain undisturbed for a long period of time (even for many years), until a stimulus "revives" them, causing the recurrence of the disease17.
Identification of the niche in which the CSCs reside in a quiescent state could represent a valid tool for patient follow-up, even after they have obtained an R0 surgical resection or after a complete response to treatment, for an early diagnosis of recurrence and metastasis18,19.
CONCLUSIONS
The results demonstrate that CSC-enriched spheroid cultures accurately acquire the characteristics of primary colorectal tumours. More importantly, CSCs lines provide an efficient experimental system for performing pre-clinical tests of chemotherapeutic and immuno-therapeutic drugs, which leads to a deeper understanding of the molecular determinants of resistance to therapy and tumour sensitivity to combination therapies20-22.
Since it has been shown that the microenvironment plays a key role in tumour development, studies into the relationship between CSC and the immune system have aimed to develop effective therapeutic strategies targeted at the ability of CSCs to avoid immune surveillance23.
The isolation of CSCs represents a valid support to surgical therapy and allows a more in-depth study of the tumour in order to be able to administer a personalised therapy that is more effective and long-term.
Our study, although conducted on a small number of patients, showed that mortality, tumour stage and grading were much higher in the group from whom stem cells were isolated (60%), compared to those in which they were not identified (11.8%).
This study seems to indicate a tight link between CSCs features and a patient's prognosis.
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