1. Volume 25, Issue 2, Pages 89-99 (June 2018)
Revisiting colorectal cancer animal model – An improved metastatic model for distal rectosigmoid colon carcinoma 
Rui Caetano-Oliveira, Marcos António Gomes, Ana Margarida Abrantes, Edgar Tavares-Silva, Marco Carvalho Oliveira, Mafalda Laranjo, Débora Basílio Queirós, João Casalta-Lopes, Salomé Pires, Lina Carvalho, Rosa Gouveia, Paulo Rodrigues Santos, Denise Gonçalves Priolli, José Guilherme Tralhão, Maria Filomena Botelho 
Pathophysiology 
Volume 25, Issue 2, Pages (June 2018)
DOI: /j.pathophys
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2. Fig. 1 Morphological aspects of the cellular lines used.
On all the cellular lines it is possible to observe cell size and shape variation, nuclear pleomorphism, hyperchromatic nuclei and multinucleation. A – C2BBe1 cell line, H&E; B – C2BBe1 cell line, Pap stain; C – WiDr cell line, H&E; D – WiDr cell line, Pap stain.
Pathophysiology  , 89-99DOI: ( /j.pathophys )
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3. Fig. 2 Immunohistochemistry studies.
A – Absence of nuclear staining in the C2BBe1 cellular line, 400x; B – Strong and diffuse staining for p53 in the WiDr cellular line, 400x; C – CD44 staining in WiDr cellular lines, 400x; D- VEGF strong staining, WiDr cells, 400x
Pathophysiology  , 89-99DOI: ( /j.pathophys )
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4. Fig. 3 Colostomy and sigmoid mucosal-cutaneous fistula.
Schematic illustration and photography of descending colostomy with a mucosal-cutaneous fistula of thesigmoid colon (A) and cecostomy (B).
Pathophysiology  , 89-99DOI: ( /j.pathophys )
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5. Fig. 4
Histological evaluation of tumor inoculated at the colorectal stoma – WiDr and C2BBe1 cellular lines, H&E stain.
Representative images of histological specimen isolated from colorectal stoma, following injection of 1 × 107 WiDr and C2BBe1 CRC cells: A – tumor developed in the colon wall, WiDr cell line, H&E 40x; B – Tumor architecture: cribriform pattern, with high number of mitosis, WiDr cell line, H&E 400x; C – neural invasion at the myenteric plexus, WiDr cell line, H&E 40x; D – tumor with mucinous micropapillary pattern, C2BBe1 cell line, H&E 20x
Pathophysiology  , 89-99DOI: ( /j.pathophys )
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6. Fig. 5
LS1034, C2BBe1 and WiDr CRC cell lines are significantly resistant to NK cell specific cytotoxicity. (A) Dot plots from flow cytometry experiments showing degranulation (% CD107a NK cells) after co-culture of CRC cell lines with non-stimulated (NS) and 24 h recombinant human IL-2 (rhIL-2) stimulation of human NK cells from healthy donors. Dot plots of NK cells gated on CD3-CD56+ lymphocyte singlets of representative donors are shown for each cell line (including K562 as reference) and condition (NS vs. rhIL-2). (B) Graphical representation of the analysis of NK cell degranulation test using 20 healthy donors. Significant decrease of sensitivity to NK cell lysis was observed for the 3 CRC cell lines when compared to K562 sensitive cell line. Although NK cell activation with rhIL-2 increase killing in the same proportions for all cell lines, CRC cell lines shown significant decrease sensitivity to NK killing. (C) Graphical representation of the IFN-γ secreting NK cells from 20 healthy donors after co-culture with CRC cell lines. The ability to induce cytokine production by NK cells is significantly reduced for all CRC cell lines resembling those results observed for the degranulation tests. Apparently, LS1034 and C2BBe1 show a significant response to rhIL-2 stimulation when compared to WiDr.
Statistical comparison of means of different cell lines and conditions was performed using two-way Mann-Whitney test and plots represent mean ± SEM values.
Pathophysiology  , 89-99DOI: ( /j.pathophys )
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7. Fig. 6
Immunohistochemical studies on the WiDr inoculated animal – distal colorectal fistula.
A – CD44 membranous staining in neural invasion, WiDr cell line, 100x; B – High VEGF expression in the main tumor periphery, WiDR cell line, 40x.
Pathophysiology  , 89-99DOI: ( /j.pathophys )
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8. Fig. 7 In-vivo molecular imaging assessment.
Static image acquired 30 min after administration of 99mTc-MIBI, 10 weeks after implantation with C2BBe1 cell line (Group B) (Left). Static images acquired 60 min after administration of 99mTc-MIBI, 6 weeks after implantation with WiDr cell line (Group A) (Right).
Pathophysiology  , 89-99DOI: ( /j.pathophys )
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