Understanding the Role of Cancer Stem Cells in Treatment Resistance and Poor Outcomes of Rectal Cancer in Indian Population

Introduction

Rectal cancer is the most diagnosed cancer and a leading cause for deaths due to cancers globally, affecting both the genders. In India the data from Indian Council of Medical Research (ICMR) has shown the rectal cancer accounted for 10.0% among all cancer cases (6, 63,000) diagnosed in the year 2013. The incidence of rectal cancer has shown an increasing trend of the disease burden.The estimated age standardised rate (ASR) of rectal cancer in India is 4.3 and 3.5 per 100,000 in males and females, respectively.¹

Recent studies suggest that intratumoral heterogeneity may contribute to therapy failure.² These heterogeneous tumour cells differ from one another in growth,metabolism, and apoptosis. Among heterogeneous tumour cells, a relatively small fraction of cells called Cancer stem cells (CSCs) is gaining importance in recent cancer research.³ The research has also shown significant genetic variation in rectal carcinoma. Failure of both chemotherapy and radiation treatment is largely attributed to stem cells due to resistance mechanisms.^4-5 Effective targeting of rectal cancer stem cells is expected to increase the success rate of treatment. ⁶ We hypothesise that understanding cancer stem cells is a more effective means of treating cancer. Additionally, understanding resistance pathways of cancer stem cells will help treat the tumour effectively and provide better understanding of residual resistant clones of rectal cancer requiring a unique approach.

Materials and Methods

Study design

• Prospective, observational, non-interventional study

Study period

• This trial aimed to include all the eligible, consenting patients presenting to the institute from March 1, 2018 to February 29, 2020.

Study outcomes

• To identity and quantify cancer stem cells in rectal cancer cell lines of Indian patients.
• To estimate overall survival outcomes of patients and its correlation to stem cell population isolation.

Inclusion criteria

• Adult patient >= 18 years of age
• Histologically confirmed rectal cancer
• Eastern Cooperative Oncology Group (ECOG) - Performance status (PS) 0 or 1
• Clinical stage T1-4/N0-2/M0 at presentation
• Patients planned for chemoradiation followed by surgery

Exclusion criteria

• Patients with more than one primary malignancy
• History of any prior treatment before consenting
• Patients with lack of understanding and those anticipated to be noncompliant to protocol

Chemicals and Materials

Cell Culture⁷

1. I or II or III or IV type collagenase. Store at 4°C
2. Hyaluronidase. Store at 4°C.
3. Phosphate Buffered Saline (PBS) without Ca2+, Mg2+. Store at 4°C.
4. 70 μm Falcon strainers. Store at room temperature.
5. Dulbecco’s modified Eagle’s medium (DMEM). Store at 4°C.
6. Foetal Bovine Serum (FBS). Store at -20°C.
7. Penicillin and Streptomycin, Pen/Strep. Store at -20°C.
8. Bovine Serum Albumin (BSA). Store at room temperature.
9. Trypsin-EDTA solution. Store at 4°C.
10. Dimethyl sulfoxide (DMSO). Store at room temperature at dark.

Antigens and Conjugates

1. CD 24 (mAb)
2. CD 44 (mAb)
3. CD 133 (mAb)
4. Oct 4 (mAb)
5. Sox 2 (polyclonal)
6. Nanog (polyclonal)

Side Population⁴

1. Hoechst 33342. Stored at 4° C.

Detailed description of the procedure

Before surgery, each patient was explained the details regarding the study and their consent was obtained the previous day or just prior to shifting to the preoperative area.From each of the rectal cancer sample, approximately 3-5 grams of tissue sample which was confirmed histopathologically for stage and grade was collected in a sterile container containing Phosphate Buffered Saline (PBS) prior to chemoradiation and complete incision of tumours after chemoradiation.

For further processing, the sample was shifted to a laboratory in sterile condition, the process of which is explained below (Figure 1)

i. For preparing single cell suspension from tumour tissue samples⁷

• The tumour sample was dissected, minced, and washed in PBS. All the visible clumps were removed.
• The cells were then filtered through a 70 μm mesh cell strainer to further dissociate the cells.
• The remains of these cells on the strainer were subjected to 2-3 mL of culture medium via pipetting and the remaining tissue along with the strainer was then discarded.
• DMEM was added in the tube to make the total volume up to 50 mL. The tube was then centrifuged at 2000 rpm.
• The supernatant was discarded, and the cells (pellet) were resuspended at 2.0×10⁶ cells/mL in prewarmed DMEM and divided into two portions.

ii. Hoechst staining for cancer stem cells⁴

• The portions were incubated in standard culture medium with 5 μg/mL Hoechst 33342 for 90 minutes at 37 degrees on a shaking bath.
• After 90 minutes, these cells were spun down in cold and then resuspended in cold PBS. To prevent the leakage of Hoechst 33342 dye, all the further proceedings were carried out at 4 degrees.
• The dye was excited at 350 nm UV; the tumour cells that take up the dye appear bright blue (Hoechstbr) while the cancer stem cells which pumps out the dye by ATP-binding cassette transporters appear dim blue (Hoechstlow).
• By carrying out cell counting, the number of each type of cell was calculated and their percentage was found.

Statistical analysis

The sample size was calculated by N-master software. For relative precision of 10% with confidence limit of 95%, with stem cell collection failure of 40%, a sample size of 24 was required. All the laboratory experiments were performed in triplicates taking at least three replicas of each sample/concentration. The data were entered in the Excel sheet and analysed using SPSS 18.0 version for calculating stem cell population percentage to overall survival on 20 December 2022. Overall survival is the time from randomization to death of any cause till the last follow up.

Results

During the study period, 91 patients were diagnosed with rectal cancer but only 12 patients who met the inclusion criteria and consented for study were considered for analysis (Table 1). The study population had ten male and two female patients in the age range of 29 to 67 years. Clinical stage at presentation for most patients was Stage 3 and one patient was pathological Stage 1 after chemoradiation (Table 1). Nine patients could give two samples i.e., before and after chemoradiation. Out of three patients who were unable to give the post chemoradiation sample, one had complete pathological response, progressive disease, and defaulted surgery.

A total of 21 rectal cancer tissue samples were collected during the study period. Within 48 hours of collection, we could prepare single cell suspension culture (SCSC) from tumour tissue samples of eight patients prior to their chemoradiation. We failed to prepare SCSC in patients who underwent surgery post chemoradiation as surgical specimens processing took more than 48 hrs for decontamination; therefore these specimens were excluded from study. The maximum duration of viability of prepared SCSC was around 10 days. Based upon the Hoechst side population’s flow cytometry technique, quantification of stem cells was based upon counting the Hoechst low stained stem cell in the cell counter (Figure 2). Range of percentage of Hoechst low stem cells to Hoechstbr tumour cells was found to be 0.2 to 0.9% in eight SCSC (Table 1).

Pearson’s Correlation Coefficient: [y=0.0143*X+0]; Pearson’s Coefficient= 0.617; p<0.033

Overall survival of the study population was assessed on 20 December 2022. We were unable to follow up with three patients after one year. Median and mean of overall survival in study participants was 41 and 37 months, respectively. Pearson’s correlation coefficient was used to compare means of t Hoechst low. Stem cell population and overall survival among the study sample was found to be statistically significant. (Pearson Correlation Coefficient= 0.61, p <0.01) (Figure 3).

Discussion

Stem cells are a kind of cells that are unspecialized in the human body. Stem cells have the capacity of differentiating to different cells of the living being and possess the potential of self-renewal. They are present at both the stages like embryo cells as well as adult cells. The extraction of induced pluripotent stem cells (iPSCs) has transformed the research and clinical trials in this area. These in comparison to embryonic stem cells (ESCs) could generate in an unlimited manner and differentiate into essentially any specialised cell type. Dissimilar to ESCs, induced pluripotent stem cells are generated from somatic cells, preventing ethical issues, and thus providing a new area for in vivo models of disease for studies of pathogenesis and drug screening and serve as a source of cells for experimental transplantation therapies.⁸ Stem cells are known to be the earliest developed cells in many body tissues; multiple mutations must accumulate in cancer. Due to this reason, the primary oncogenic mutations must have occurred in stem cells.² Among heterogeneous tumour cells, CSCs are gaining importance due to significant characteristics of enhanced tumorigenicity, capacity for self-renewal and differentiation, and resistance to radiation and chemotherapy which is responsible for poor outcomes in cancer patients.^9,10

Rectal cancer is one of the challenging cancers to treat due to its location in the pelvis close to complex genitourinary organs. Patients with localised rectal cancer undergo chemo-radiation with 5 FU or capecitabine followed by surgery with adjuvant Oxaliplatin based combination chemotherapy with either 5 FU infusion or capecitabine tablets or giving preoperative chemotherapy to improve historical outcomes.¹¹ CSCs are attributed for resistance to chemoradiation, which can be identified by CSCrelated biomarkers like CD133+, CD44, ALDH1, SOX2 and OCT4.^9,12 The Hoechst side population’s flow cytometry technique was used to obtain rectal cancer stem cells based on the dye efflux properties of the ATPbinding cassette (ABC) transporters due to the presence of stemness genes.¹³

In our study, we could isolate stem cell population from eight patients which showed positive correlation to overall survival despite smaller sample size. Based on preliminary data, the protocols can be optimized for production of single cell cultures to facilitate confirmation and quantification of stem cell population cells by CSCrelated biomarkers like CD133+, CD44, and CD44 by cell sorter and molecular pathways in future studies for assessing effectiveness of multimodality treatments like previous studies.^9,12,14-16 In further studies, rectal cancer organoid platform instead of animal models would be ideal and allows us to bypass our limitation of failure to isolate stem cell population from chemoradiation samples.^17-19

Conclusion

Cancer stem cell populations in rectal cancer patients can be attributed to poor outcomes owing to the resistance mechanisms to chemoradiation, termed as stemness. By better understanding the stemness and targeting of pathways by novel therapies, we can improve current poor outcomes in operable rectal cancers undergoing chemoradiation

Conflict of interest

Nil

Acknowledgements

We want to thank RGHUS for Grant 17M043 as research project for the year 2017-18 and Dr Nandakumar, DRP, Ramaiah Medical College for Statistical analysis.