The study aimed to analyze the molecular profiles of surgical margins and colorectal cancer (CRC) tumors to identify prognostic markers. The results showed that cancer stem-like cells (CSCs) were present in 84.84% of tumors but also enriched in the distal surgical margin in 63.63% of cases. CSCs in distal margins correlated with shorter resection lengths, with 81.8% of margins under 2cm positive for CSCs compared to 0% over 5cm. Additionally, genes related to drug resistance and stemness were overexpressed in distal margins versus normal tissue. While CSC presence did not impact survival rates, the results suggest CSCs in surgical margins may drive tumor recurrence and wider margins could help reduce recurrence risk

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

2. 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.

3. 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%)

4. 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%)

5. 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.

6. 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

7. 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%

8. 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

9. 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.

10. 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

11. 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.

12. 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].