“Colorectal cancer stem cells in resection margins: Are they drivers of

tumourigenesis

Introduction

Aim of our study:-• To dissect the molecular profile of surgical margins and CRC tumors and

use this knowledge for developing a set of prognostic markers that could be used for clinical intervention.

• Validating the existing molecular markers to identify cancer stem like cells in CRC tumor and surgical margins and using this knowledge to study the tumor heterogeneity and its relevance in local recurrence, distant metastasis and drug resistance property of CRC tumor.

Pretreatment CEA 2.9 (.03-41000)ng/ml

Pre-Operative Chemotherapy

Yes 19(57.6%)

No 14 (42.4%)

Preoperative Radiotherapy

Yes 20(60.6%)

No 13(39.4%)

Demography

Age 54.7 (27-75)

Sex Male 17(51.5%)

Female 16(48.5%)

Symptoms

Bleeding PR 28(84.8%)

Duration 108days(10-360)

Site of Disease Rectum 26(78.8%)

Colon 7(21.2%)

Post treatment TNM stage

0 2 (6.06%)

1 7(21.21%)

IIA 8(24.24%)

IIIA 2(6.06%)

IIIB 11(33.33%)

IIIC 1(3.03%)

IVA 2(6.06%)

Distal margin 3.75 (0-13) cms

Proximal Margin 13.92(4-22)cms

No of Lymph nodes dissected 8.5(0-28)

No of metastatic lymph nodes 1.2(0-5)

Stem Cell Like Cells at Tumour 28(84.84%)

Stem Cell Like Cells at Margin 21(63.63%)

Relevance of CSC marker in clinical scenario

Length of resected distal margins(cm)

Negative SP Positive SP Total no of patients

0-2 2(18.18%) 9(81.8%) 11

2-5 7(36.8%) 12(63.2%) 19

5-10 3(100%) 0 3

Side population analysis CSC in distal margins reveals that a minimal resection length of 5cm would suffice to remove residual tumor cells.

RESULTS Molecular analysis of surgical margins

Real time PCR and western blot assay show that surgical margins are enriched with markers related to drug resistance ,stemness and metastasis

Spatial distribution of CSC in margins of CRC

FACS based quantification of Cancer stem like cells clearly shows that CD133 positive cells are enriched in distal surgical margin while CD44 positive cells are enriched in circumferential margin and adjacent lymphnodes.

Since circumferential margin and lymnphnodes are implicated in distant metastasis we hypothesize that CD133 positive cells might be important in maintenance of primary tumor while CD44+ve cells might be critical in initiating distant metastasis.

Margins CD133 positive CSC cells

CD44 Positive CSC cells

Normal 1.8% 3.2%

Tumor 8.9% 6.5%

Proximal 2.1% 2%

Distal 15.9% 3.5%

Circumferential 3.0% 8.4%

Lymphnode 5.0% 14.2%

Imaging Cancer Stem like 5fu resistant cells in Rectal Tumor and Distal surgical margin

Normal Rectum Rectal Tumor Distal surgical margin

IHC imaging of CRC tumors reiterates the above observation

Cells stained in green color overexpress drug efflux proteins which are critical in drug resistance phenomenon

Margins CD133+CD44 CELLS CD31 CELLS

NORMAL 0.8% 0.5%

TUMOR 38.4% 27.3%

DISTAL 2.1% 1.4%

PROXIMAL 1% 0.6%

LYMPH NODE 1.2% 8.9%

Endothelial cells can act as a potential niche for cancer stem like cells in Rectal Cancer

Multiparametric FACS analysis clearly shows that presence of cancer stem like cells is positively correlated with presence of CD31 +ve endothelial cells in the same region. Hence a highly vascularized tumor should be a cause of concern as it would have a higher amount of residual tumor cells.

Stem Cells at Tumour p value

No Yes

DFS 87.5% 66.9% 0.1663

OS 71.4% 73% 0.3718

Stem Cells at Margin

DFS 68.6% 77.8% 0.3897

OS 79.6% 68.2% 0.2103

Survival probability at 3 years

Result The CSC was present at tumour in 28 out of 33 (84.84%)patients, at surgical

margin in 21 out of 33 (63.63%) patients and in normal mucosa in 5 out of 33 (15.15%)patients

CSC at margin were positive in 9 out of 11 (81.8%) when distal margin was less than 2cms, 12 out of 19(63.2%) when it was 2cms-5cms and none in three cases (0%) when distal margin was more than 5cms (correlation -0.405 pvalue .033)

But there was no correlation of percentage of ‘stem cell like cells’ at tumour or margin with location of tumour, serum CEA level, stage of disease, grade, previous therapy, lymph node metastasis.

In parallel to flow cytometry based assays, we also performed an immuno-histochemical analysis and we found an over-expression of drug resistant genes such as ABCG2, MDR, MRP1 in distal surgical margin compared to normal and tumor tissues. Further analysis using western blot and quantitative real time PCR proved this observation.

During the study period there was no difference in survival probability between groups with or without tumour stem cell like cells at tumour or surgical margin.

Result This is the first study which has focused in understanding CSCs distribution in surgical

margins of colorectal cancer. Interestingly we observed an enrichment of CSCs in distal surgical margin and this enrichment was 5 to 10 fold higher than the tumor itself.

In accordance with the role of CSCs in drug resistance we observed an increased expression of ABC transporters in tumor and distal surgical margins compared to adjacent normal tissue. This further strengthens our hypothesis that distal surgical margin serves as a right niche for colorectal cancer stem cells.

This finding is highly relevant for clinical setting as distal margins greater than 5cm had almost no CSCs, hence whenever possible a wider surgical margin might reduce the recurrence rate considerably.

With these observations we are moving ahead in our pursuit to isolate CSCs and enrich them in invitro culture conditions. These enriched CSC cultures will be treated with various factors from distal, proximal and circumferential margins and then studied for their tumorigenic and drug resistant properties in an orthotopic SCID mice model[5].