Figure 1 Results of tumorsphere formation assay

Results of tumorsphere formation assay showing the absence of spheroidal cellular aggregates (tumorspheres) in normal skin cell line (A) and the presence of tumorspheres in skin papilloma (B, papilloma 01), fibropapilloma (C, papilloma 02 and D, papilloma 03) and esophageal carcinoma cell lines (E and F). Total magnification of 100 X. Scale bar of 100 µm.

Figure 2 Quantitative analysis of Oct-3/4 transcription factor by flow cytometry

A) skin papilloma cells without any antibody; B) skin papilloma cells incubated with secondary antibody Alexa Fluor 488, showing absence of unspecific labelling; Oct-3/4 expression levels in: normal skin (C, BPV-free), skin papilloma (D, papilloma 01), fibropapilloma (E, papilloma 02 and F, papilloma 03) and G) esophageal carcinoma cell lines. H) Histogram based on the cell percentage expressing the Oct-3/4 transcription factor. Results show an increase in Oct-3/4 expression levels in BPV-infected cell lines. Cell analyzed in second passage (P2). Total of 10,000 events analyzed.

Figure 3 Immunodetection of Oct-3/4 transcription factor

Immunodetection of Oct-3/4 transcription factor showing the increase of fluorescence signal in skin papilloma (papilloma 01), fibropapilloma (papilloma 02 and 03) and esophageal carcinoma cell lines. Results also show the nuclear labelling of Oct-3/4 in esophageal carcinoma cells. Images obtained with total magnification of 400X, scale bar of 100 µm.

Figure 4 Morphological analysis by phase contrast analysis

Phase contrast analysis showing the maintenance of cell polarity along the six passages of normal skin cell line (BPV-free) and the loss of cell polarity in all passages analyzed of skin papilloma (papilloma 01), fibropapilloma (papilloma 02 and 03) and esophageal carcinoma cell lines. All BPV-infected cell lines show the presence of lamellipodia and filopodia, suggesting the acquisition of migratory phenotype. Imagens obtained in total magnification of 100X, scale bar of 100 µm.

Figure 5Diagnostic odds ratio of CLSM in the diagnosis of (a) Malignant Melanoma and (b) Basal Cell Carcinoma.

A) BPV-free normal skin, B) cutaneous papilloma, C) firbopapilloma 01, D) fibropapilloma 02, E and F) esophageal carcinoma. Results show the F-actin labelling in plasmatic membrane, reinforcing the presence of filopodia in all primary cultures cells derived from BPV-infected neoplasm.

Figure 6 Screen electron-micrograph (SEM)

A)keratinocyte-like of normal skin cell line, presenting apical-basal polarity (6664 X), B)fibroblast-like of normal skin cell line, showing fusiform morphology (8280 X), cells of papilloma 01 (C, 9829 X, skin papilloma), 02 (D, 9388 X, fibropapilloma), 03 (E, 2893 X, fibropapilloma), and esophageal carcinoma (F, 2898 X) cell lines showing loss of polarity, extensive lamellipodia, and abundant filopodia, G) lamellipodia site, with filopodia, observed in esophageal carcinoma cell line (53021 X), inter-cytoplasmic bridges observed in papilloma 02 (H, 3210 X), and esophageal carcinoma cell lines (I, 25777 X).

Figure 7 Results of cell migration assay by time-lapse video microscope

Results of cell migration assay by time-lapse video microscope showing the average speed in µm/minute in 80 time-point, in a total of 20 hours of analysis: A) normal skin, B) papilloma 01 (skin papilloma), C) papilloma 02 (fibropapilloma), D) papilloma 03 (fibropapilloma) and E) esophageal carcinoma cell lines. F) Histogram based on mean of cell velocity along the 80 time-point analyzed, followed by the standard deviation (bars) demonstrating the highest migration velocity in carcinoma cell line. Papilloma 01 and 03 show intermediated migration velocity, as confirmed by the Tukey’s test. Normal skin and papilloma 02 did not reveal statistical difference between their migration velocity.

 

Passage 1

Passage 2

Passage 3

Passage 4

 

Passage 5

Passage 6

Normal skin

-

-

-

-

-

-

Papilloma 01

BPV-1

BPV-1 and 2

BPV-2

BPV-1 and 2

 

BPV-1 and 2

-

Papilloma 02

BPV-1, 2 and 4

BPV-1, 2 and 4

BPV-2 and 4

BPV-1 and 2

 

BPV-1, 2 and 4

BPV-2 and 4

Papilloma 03

BPV-2 and 4

BPV-1 and 2

BPV-2

BPV-2 and 4

 

BPV-1 and 2

BPV-2

Carcinoma

BPV-1, 2 and 4

BPV-1, 2 and 4

BPV-1, 2 and 4

BPV-1, 2 and 4

 

BPV-1, 2 and 4

BPV-1, 2 and 4

Table1 BPV types identified in primary cell cultures

Results based on Araldi et al. [7]

 

Multiple comparison

Mean Difference

q

P< 0.05

 

Normal skin and papilloma 01

-0.02171

2.818

No

 

Normal skin and papilloma 02 (FP)

0.004682

0.608

No

 

Normal skin and papilloma 03 (FP)

-0.04973

6.457

Yes

 

Normal skin and esophageal carcinoma

-0.06675

8.666

Yes

 

Papilloma 01 and Papilloma 02 (FP)

0.02639

3.426

No

 

Papilloma 01 and Papilloma 03 (FP)

-0.02803

3.639

No

 

Papilloma 01 and esophageal carcinoma

-0.04504

5.848

Yes

 

Papilloma 02 (FP) and papilloma 03 (FP)

-0.05441

7.065

Yes

 

Papilloma 02 (FP) and esophageal carcinoma

-0.07143

9.274

Yes

 

Papilloma 03 (FP) and esophageal carcinoma

-0.01702

2.209

No

Table 2Result of Tukey’s multiple comparison test

FP - fibropapilloma